diff --git a/packages/vite/src/node/config.ts b/packages/vite/src/node/config.ts
index 7b4107c8aafe05..1933a5484b7565 100644
--- a/packages/vite/src/node/config.ts
+++ b/packages/vite/src/node/config.ts
@@ -2,6 +2,7 @@ import fs from 'node:fs'
 import fsp from 'node:fs/promises'
 import path from 'node:path'
 import { pathToFileURL } from 'node:url'
+import { promisify } from 'node:util'
 import { performance } from 'node:perf_hooks'
 import { createRequire } from 'node:module'
 import colors from 'picocolors'
@@ -69,6 +70,7 @@ import type { ResolvedSSROptions, SSROptions } from './ssr'
 import { resolveSSROptions } from './ssr'
 
 const debug = createDebugger('vite:config')
+const promisifiedRealpath = promisify(fs.realpath)
 
 export type {
   RenderBuiltAssetUrl,
@@ -1106,7 +1108,11 @@ async function loadConfigFromBundledFile(
   // for cjs, we can register a custom loader via `_require.extensions`
   else {
     const extension = path.extname(fileName)
-    const realFileName = await fsp.realpath(fileName)
+    // We don't use fsp.realpath() here because it has the same behaviour as
+    // fs.realpath.native. On some Windows systems, it returns uppercase volume
+    // letters (e.g. "C:\") while the Node.js loader uses lowercase volume letters.
+    // See https://github.com/vitejs/vite/issues/12923
+    const realFileName = await promisifiedRealpath(fileName)
     const loaderExt = extension in _require.extensions ? extension : '.js'
     const defaultLoader = _require.extensions[loaderExt]!
     _require.extensions[loaderExt] = (module: NodeModule, filename: string) => {