diff --git a/packages/vite/src/node/server/index.ts b/packages/vite/src/node/server/index.ts
index 67b455735..9e81484c5 100644
--- a/packages/vite/src/node/server/index.ts
+++ b/packages/vite/src/node/server/index.ts
@@ -509,7 +509,10 @@ export async function _createServer(
     _importGlobMap: new Map(),
     _forceOptimizeOnRestart: false,
     _pendingRequests: new Map(),
-    _fsDenyGlob: picomatch(config.server.fs.deny, { matchBase: true }),
+    _fsDenyGlob: picomatch(config.server.fs.deny, {
+      matchBase: true,
+      nocase: true,
+    }),
     _shortcutsOptions: undefined,
   }
 
diff --git a/playground/fs-serve/__tests__/base/fs-serve-base.spec.ts b/playground/fs-serve/__tests__/base/fs-serve-base.spec.ts
index 4660fafcc..51e87ccd3 100644
--- a/playground/fs-serve/__tests__/base/fs-serve-base.spec.ts
+++ b/playground/fs-serve/__tests__/base/fs-serve-base.spec.ts
@@ -92,7 +92,13 @@ describe.runIf(isServe)('main', () => {
   })
 
   test('denied', async () => {
-    expect(await page.textContent('.unsafe-dotenv')).toBe('404')
+    expect(await page.textContent('.unsafe-dotenv')).toBe('403')
+  })
+
+  test('denied EnV casing', async () => {
+    // It is 403 in case insensitive system, 404 in others
+    const code = await page.textContent('.unsafe-dotEnV-casing')
+    expect(code === '403' || code === '404').toBeTruthy()
   })
 })
 
diff --git a/playground/fs-serve/__tests__/fs-serve.spec.ts b/playground/fs-serve/__tests__/fs-serve.spec.ts
index 86e030326..9d9d4c6ec 100644
--- a/playground/fs-serve/__tests__/fs-serve.spec.ts
+++ b/playground/fs-serve/__tests__/fs-serve.spec.ts
@@ -92,7 +92,13 @@ describe.runIf(isServe)('main', () => {
   })
 
   test('denied', async () => {
-    expect(await page.textContent('.unsafe-dotenv')).toBe('404')
+    expect(await page.textContent('.unsafe-dotenv')).toBe('403')
+  })
+
+  test('denied EnV casing', async () => {
+    // It is 403 in case insensitive system, 404 in others
+    const code = await page.textContent('.unsafe-dotEnV-casing')
+    expect(code === '403' || code === '404').toBeTruthy()
   })
 })
 
diff --git a/playground/fs-serve/root/src/index.html b/playground/fs-serve/root/src/index.html
index 5de6804a7..06bee3f86 100644
--- a/playground/fs-serve/root/src/index.html
+++ b/playground/fs-serve/root/src/index.html
@@ -45,6 +45,7 @@
 
 <h2>Denied</h2>
 <pre class="unsafe-dotenv"></pre>
+<pre class="unsafe-dotEnV-casing"></pre>
 
 <script type="module">
   import '../../entry'
@@ -236,7 +237,9 @@
     })
 
   // .env, denied by default
-  fetch(joinUrlSegments(base, joinUrlSegments('/@fs/', ROOT) + '/root/.env'))
+  fetch(
+    joinUrlSegments(base, joinUrlSegments('/@fs/', ROOT) + '/root/src/.env'),
+  )
     .then((r) => {
       text('.unsafe-dotenv', r.status)
     })
@@ -244,6 +247,17 @@
       console.error(e)
     })
 
+  // .env, for case insensitive file systems
+  fetch(
+    joinUrlSegments(base, joinUrlSegments('/@fs/', ROOT) + '/root/src/.EnV'),
+  )
+    .then((r) => {
+      text('.unsafe-dotEnV-casing', r.status)
+    })
+    .catch((e) => {
+      console.error(e)
+    })
+
   function text(sel, text) {
     document.querySelector(sel).textContent = text
   }