diff --git a/crates/uv-cli/src/lib.rs b/crates/uv-cli/src/lib.rs
index 26d9f3ff501a..b8722cba3ffb 100644
--- a/crates/uv-cli/src/lib.rs
+++ b/crates/uv-cli/src/lib.rs
@@ -2633,9 +2633,12 @@ pub struct RunArgs {
 
     /// Load environment variables from a `.env` file.
     ///
+    /// Can be provided multiple times, with subsequent files overriding values defined in
+    /// previous files.
+    ///
     /// Defaults to reading `.env` in the current working directory.
-    #[arg(long, value_parser = parse_file_path, env = EnvVars::UV_ENV_FILE)]
-    pub env_file: Option<PathBuf>,
+    #[arg(long, env = EnvVars::UV_ENV_FILE)]
+    pub env_file: Vec<PathBuf>,
 
     /// Avoid reading environment variables from a `.env` file.
     #[arg(long, conflicts_with = "env_file", value_parser = clap::builder::BoolishValueParser::new(), env = EnvVars::UV_NO_ENV_FILE)]
diff --git a/crates/uv/src/commands/project/run.rs b/crates/uv/src/commands/project/run.rs
index d98171c70aba..23b6aa48ecf2 100644
--- a/crates/uv/src/commands/project/run.rs
+++ b/crates/uv/src/commands/project/run.rs
@@ -8,7 +8,7 @@ use std::path::{Path, PathBuf};
 use anstream::eprint;
 use anyhow::{anyhow, bail, Context};
 use futures::StreamExt;
-use itertools::Itertools;
+use itertools::{Either, Itertools};
 use owo_colors::OwoColorize;
 use tokio::process::Command;
 use tracing::{debug, warn};
@@ -79,7 +79,7 @@ pub(crate) async fn run(
     native_tls: bool,
     cache: &Cache,
     printer: Printer,
-    env_file: Option<PathBuf>,
+    env_file: Vec<PathBuf>,
     no_env_file: bool,
 ) -> anyhow::Result<ExitStatus> {
     // These cases seem quite complex because (in theory) they should change the "current package".
@@ -109,52 +109,58 @@ pub(crate) async fn run(
 
     // Read from the `.env` file, if necessary.
     if !no_env_file {
-        let env_file_path = env_file.as_deref().unwrap_or_else(|| Path::new(".env"));
-        match dotenvy::from_path(env_file_path) {
-            Err(dotenvy::Error::Io(err)) if err.kind() == std::io::ErrorKind::NotFound => {
-                if env_file.is_none() {
-                    debug!(
-                        "No environment file found at: `{}`",
-                        env_file_path.simplified_display()
+        let env_file_paths = if env_file.is_empty() {
+            Either::Left(std::iter::once(Path::new(".env")))
+        } else {
+            Either::Right(env_file.iter().rev().map(PathBuf::as_path))
+        };
+        for env_file_path in env_file_paths {
+            match dotenvy::from_path(env_file_path) {
+                Err(dotenvy::Error::Io(err)) if err.kind() == std::io::ErrorKind::NotFound => {
+                    if env_file.is_empty() {
+                        debug!(
+                            "No environment file found at: `{}`",
+                            env_file_path.simplified_display()
+                        );
+                    } else {
+                        bail!(
+                            "No environment file found at: `{}`",
+                            env_file_path.simplified_display()
+                        );
+                    }
+                }
+                Err(dotenvy::Error::Io(err)) => {
+                    if env_file.is_empty() {
+                        debug!(
+                            "Failed to read environment file `{}`: {err}",
+                            env_file_path.simplified_display()
+                        );
+                    } else {
+                        bail!(
+                            "Failed to read environment file `{}`: {err}",
+                            env_file_path.simplified_display()
+                        );
+                    }
+                }
+                Err(dotenvy::Error::LineParse(content, position)) => {
+                    warn_user!(
+                        "Failed to parse environment file `{}` at position {position}: {content}",
+                        env_file_path.simplified_display(),
                     );
-                } else {
-                    bail!(
-                        "No environment file found at: `{}`",
-                        env_file_path.simplified_display()
+                }
+                Err(err) => {
+                    warn_user!(
+                        "Failed to parse environment file `{}`: {err}",
+                        env_file_path.simplified_display(),
                     );
                 }
-            }
-            Err(dotenvy::Error::Io(err)) => {
-                if env_file.is_none() {
+                Ok(()) => {
                     debug!(
-                        "Failed to read environment file `{}`: {err}",
-                        env_file_path.simplified_display()
-                    );
-                } else {
-                    bail!(
-                        "Failed to read environment file `{}`: {err}",
+                        "Read environment file at: `{}`",
                         env_file_path.simplified_display()
                     );
                 }
             }
-            Err(dotenvy::Error::LineParse(content, position)) => {
-                warn_user!(
-                    "Failed to parse environment file `{}` at position {position}: {content}",
-                    env_file_path.simplified_display(),
-                );
-            }
-            Err(err) => {
-                warn_user!(
-                    "Failed to parse environment file `{}`: {err}",
-                    env_file_path.simplified_display(),
-                );
-            }
-            Ok(()) => {
-                debug!(
-                    "Read environment file at: `{}`",
-                    env_file_path.simplified_display()
-                );
-            }
         }
     }
 
diff --git a/crates/uv/src/settings.rs b/crates/uv/src/settings.rs
index a022f039ae3c..3954844e48ed 100644
--- a/crates/uv/src/settings.rs
+++ b/crates/uv/src/settings.rs
@@ -244,7 +244,7 @@ pub(crate) struct RunSettings {
     pub(crate) python: Option<String>,
     pub(crate) refresh: Refresh,
     pub(crate) settings: ResolverInstallerSettings,
-    pub(crate) env_file: Option<PathBuf>,
+    pub(crate) env_file: Vec<PathBuf>,
     pub(crate) no_env_file: bool,
 }
 
diff --git a/crates/uv/tests/it/run.rs b/crates/uv/tests/it/run.rs
index 9f8742aecaf7..a1d963c2af39 100644
--- a/crates/uv/tests/it/run.rs
+++ b/crates/uv/tests/it/run.rs
@@ -2373,9 +2373,7 @@ fn run_stdin_with_pep723() -> Result<()> {
        "#
     })?;
 
-    let mut command = context.run();
-    let command_with_args = command.stdin(std::fs::File::open(test_script)?).arg("-");
-    uv_snapshot!(context.filters(), command_with_args, @r###"
+    uv_snapshot!(context.filters(), context.run().stdin(std::fs::File::open(test_script)?).arg("-"), @r###"
     success: true
     exit_code: 0
     ----- stdout -----
@@ -2396,8 +2394,7 @@ fn run_stdin_with_pep723() -> Result<()> {
 fn run_with_env() -> Result<()> {
     let context = TestContext::new("3.12");
 
-    let test_script = context.temp_dir.child("test.py");
-    test_script.write_str(indoc! { "
+    context.temp_dir.child("test.py").write_str(indoc! { "
         import os
         print(os.environ.get('THE_EMPIRE_VARIABLE'))
         print(os.environ.get('REBEL_1'))
@@ -2406,8 +2403,7 @@ fn run_with_env() -> Result<()> {
        "
     })?;
 
-    let env_file = context.temp_dir.child(".env");
-    env_file.write_str(indoc! { "
+    context.temp_dir.child(".env").write_str(indoc! { "
         THE_EMPIRE_VARIABLE=palpatine
         REBEL_1=leia_organa
         REBEL_2=obi_wan_kenobi
@@ -2415,10 +2411,7 @@ fn run_with_env() -> Result<()> {
        "
     })?;
 
-    let mut command = context.run();
-    let command_with_args = command.arg("test.py");
-
-    uv_snapshot!(context.filters(), command_with_args,@r###"
+    uv_snapshot!(context.filters(), context.run().arg("test.py"), @r###"
     success: true
     exit_code: 0
     ----- stdout -----
@@ -2434,11 +2427,10 @@ fn run_with_env() -> Result<()> {
 }
 
 #[test]
-fn run_with_parent_env() -> Result<()> {
+fn run_with_env_file() -> Result<()> {
     let context = TestContext::new("3.12");
 
-    let test_script = context.temp_dir.child("test").child("test.py");
-    test_script.write_str(indoc! { "
+    context.temp_dir.child("test.py").write_str(indoc! { "
         import os
         print(os.environ.get('THE_EMPIRE_VARIABLE'))
         print(os.environ.get('REBEL_1'))
@@ -2447,8 +2439,7 @@ fn run_with_parent_env() -> Result<()> {
        "
     })?;
 
-    let env_file = context.temp_dir.child(".env");
-    env_file.write_str(indoc! { "
+    context.temp_dir.child(".file").write_str(indoc! { "
         THE_EMPIRE_VARIABLE=palpatine
         REBEL_1=leia_organa
         REBEL_2=obi_wan_kenobi
@@ -2456,19 +2447,14 @@ fn run_with_parent_env() -> Result<()> {
        "
     })?;
 
-    let mut command = context.run();
-    let command_with_args = command
-        .arg("test.py")
-        .current_dir(context.temp_dir.child("test"));
-
-    uv_snapshot!(context.filters(), command_with_args,@r###"
+    uv_snapshot!(context.filters(), context.run().arg("--env-file").arg(".file").arg("test.py"), @r###"
     success: true
     exit_code: 0
     ----- stdout -----
-    None
-    None
-    None
-    None
+    palpatine
+    leia_organa
+    obi_wan_kenobi
+    C3PO
 
     ----- stderr -----
     "###);
@@ -2477,100 +2463,143 @@ fn run_with_parent_env() -> Result<()> {
 }
 
 #[test]
-fn run_with_env_omitted() -> Result<()> {
+fn run_with_multiple_env_files() -> Result<()> {
     let context = TestContext::new("3.12");
 
-    let test_script = context.temp_dir.child("test.py");
-    test_script.write_str(indoc! { "
+    context.temp_dir.child("test.py").write_str(indoc! { "
         import os
         print(os.environ.get('THE_EMPIRE_VARIABLE'))
+        print(os.environ.get('REBEL_1'))
+        print(os.environ.get('REBEL_2'))
        "
     })?;
 
-    let env_file = context.temp_dir.child(".env");
-    env_file.write_str(indoc! { "
+    context.temp_dir.child(".env1").write_str(indoc! { "
         THE_EMPIRE_VARIABLE=palpatine
+        REBEL_1=leia_organa
        "
     })?;
 
-    let mut command = context.run();
-    let command_with_args = command.arg("--no-env-file").arg("test.py");
+    context.temp_dir.child(".env2").write_str(indoc! { "
+        THE_EMPIRE_VARIABLE=palpatine
+        REBEL_1=obi_wan_kenobi
+        REBEL_2=C3PO
+       "
+    })?;
 
-    uv_snapshot!(context.filters(), command_with_args,@r###"
+    uv_snapshot!(context.filters(), context.run().arg("--env-file").arg(".env1").arg("--env-file").arg(".env2").arg("test.py"), @r###"
     success: true
     exit_code: 0
     ----- stdout -----
-    None
+    palpatine
+    obi_wan_kenobi
+    C3PO
 
     ----- stderr -----
     "###);
 
+    uv_snapshot!(context.filters(), context.run().arg("test.py").env("UV_ENV_FILE", ".env1 .env2"), @r###"
+    success: false
+    exit_code: 2
+    ----- stdout -----
+
+    ----- stderr -----
+    error: No environment file found at: `.env1 .env2`
+    "###);
+
     Ok(())
 }
 
 #[test]
-fn run_with_malformed_env() -> Result<()> {
+fn run_with_env_omitted() -> Result<()> {
     let context = TestContext::new("3.12");
 
-    let test_script = context.temp_dir.child("test.py");
-    test_script.write_str(indoc! { "
+    context.temp_dir.child("test.py").write_str(indoc! { "
         import os
         print(os.environ.get('THE_EMPIRE_VARIABLE'))
        "
     })?;
 
-    let env_file = context.temp_dir.child(".env");
-    env_file.write_str(indoc! { "
-        THE_^EMPIRE_VARIABLE=darth_vader
+    context.temp_dir.child(".env").write_str(indoc! { "
+        THE_EMPIRE_VARIABLE=palpatine
        "
     })?;
 
-    let mut command = context.run();
-    let command_with_args = command.arg("test.py");
-
-    uv_snapshot!(context.filters(), command_with_args,@r###"
+    uv_snapshot!(context.filters(), context.run().arg("--no-env-file").arg("test.py"), @r###"
     success: true
     exit_code: 0
     ----- stdout -----
     None
 
     ----- stderr -----
-    warning: Failed to parse environment file `.env` at position 4: THE_^EMPIRE_VARIABLE=darth_vader
     "###);
 
     Ok(())
 }
 
 #[test]
-fn run_with_specific_env_file() -> Result<()> {
+fn run_with_parent_env() -> Result<()> {
     let context = TestContext::new("3.12");
 
-    let test_script = context.temp_dir.child("test.py");
-    test_script.write_str(indoc! { "
+    context
+        .temp_dir
+        .child("test")
+        .child("test.py")
+        .write_str(indoc! { "
         import os
         print(os.environ.get('THE_EMPIRE_VARIABLE'))
+        print(os.environ.get('REBEL_1'))
+        print(os.environ.get('REBEL_2'))
+        print(os.environ.get('REBEL_3'))
+       "
+        })?;
+
+    context.temp_dir.child(".env").write_str(indoc! { "
+        THE_EMPIRE_VARIABLE=palpatine
+        REBEL_1=leia_organa
+        REBEL_2=obi_wan_kenobi
+        REBEL_3=C3PO
        "
     })?;
 
-    let env_file = context.temp_dir.child(".env.development");
-    env_file.write_str(indoc! { "
-        THE_EMPIRE_VARIABLE=sidious
+    uv_snapshot!(context.filters(), context.run().arg("test.py").current_dir(context.temp_dir.child("test")), @r###"
+    success: true
+    exit_code: 0
+    ----- stdout -----
+    None
+    None
+    None
+    None
+
+    ----- stderr -----
+    "###);
+
+    Ok(())
+}
+
+#[test]
+fn run_with_malformed_env() -> Result<()> {
+    let context = TestContext::new("3.12");
+
+    context.temp_dir.child("test.py").write_str(indoc! { "
+        import os
+        print(os.environ.get('THE_EMPIRE_VARIABLE'))
        "
     })?;
 
-    let mut command = context.run();
-    let command_with_args = command
-        .arg("--env-file")
-        .arg(".env.development")
-        .arg("test.py");
+    context.temp_dir.child(".env").write_str(indoc! { "
+        THE_^EMPIRE_VARIABLE=darth_vader
+       "
+    })?;
 
-    uv_snapshot!(context.filters(), command_with_args,@r###"
+    uv_snapshot!(context.filters(), context.run().arg("test.py"), @r###"
     success: true
     exit_code: 0
     ----- stdout -----
-    sidious
+    None
 
     ----- stderr -----
+    warning: Failed to parse environment file `.env` at position 4: THE_^EMPIRE_VARIABLE=darth_vader
     "###);
 
     Ok(())
@@ -2580,26 +2609,19 @@ fn run_with_specific_env_file() -> Result<()> {
 fn run_with_not_existing_env_file() -> Result<()> {
     let context = TestContext::new("3.12");
 
-    let test_script = context.temp_dir.child("test.py");
-    test_script.write_str(indoc! { "
+    context.temp_dir.child("test.py").write_str(indoc! { "
         import os
         print(os.environ.get('THE_EMPIRE_VARIABLE'))
        "
     })?;
 
-    let mut command = context.run();
-    let command_with_args = command
-        .arg("--env-file")
-        .arg(".env.development")
-        .arg("test.py");
-
     let mut filters = context.filters();
     filters.push((
         r"(?m)^error: Failed to read environment file `.env.development`: .*$",
         "error: Failed to read environment file `.env.development`: [ERR]",
     ));
 
-    uv_snapshot!(filters, command_with_args,@r###"
+    uv_snapshot!(filters, context.run().arg("--env-file").arg(".env.development").arg("test.py"), @r###"
     success: false
     exit_code: 2
     ----- stdout -----
diff --git a/docs/configuration/files.md b/docs/configuration/files.md
index de78f91436f7..86152bffd278 100644
--- a/docs/configuration/files.md
+++ b/docs/configuration/files.md
@@ -78,11 +78,18 @@ By default, `uv run` will load environment variables from a `.env` file in the c
 directory, following the discovery and parsing rules of the
 [`dotenvy`](https://github.com/allan2/dotenvy) crate.
 
-If the same variable is defined in the environment and in the file, the value from the environment
-will take precedence.
+To load a `.env` file from a dedicated location, set the `UV_ENV_FILE` environment variable, or pass
+the `--env-file` flag to `uv run`.
 
-To disable this behavior, set `UV_NO_ENV_FILE=1` in the environment, or pass the `--no-env-file`
-flag to `uv run`.
+The `--env-file` flag can be provided multiple times, with subsequent files overriding values
+defined in previous files. To provide multiple files via the `UV_ENV_FILE` environment variable,
+separate the paths with a space (e.g., `UV_ENV_FILE="/path/to/file1 /path/to/file2"`).
+
+To disable this behavior, set the `UV_NO_ENV_FILE` environment variable to `1`, or pass the
+`--no-env-file` flag to `uv run`.
+
+If the same variable is defined in the environment and in a `.env` file, the value from the
+environment will take precedence.
 
 ## Configuring the pip interface
 
diff --git a/docs/reference/cli.md b/docs/reference/cli.md
index 873676073157..6ead947353c0 100644
--- a/docs/reference/cli.md
+++ b/docs/reference/cli.md
@@ -135,6 +135,8 @@ uv run [OPTIONS] [COMMAND]
 
 </dd><dt><code>--env-file</code> <i>env-file</i></dt><dd><p>Load environment variables from a <code>.env</code> file.</p>
 
+<p>Can be provided multiple times, with subsequent files overriding values defined in previous files.</p>
+
 <p>Defaults to reading <code>.env</code> in the current working directory.</p>
 
 <p>May also be set with the <code>UV_ENV_FILE</code> environment variable.</p>