diff --git a/Lib/asyncio/__main__.py b/Lib/asyncio/__main__.py
index 89d456b6858c07..7b0d544d59e244 100644
--- a/Lib/asyncio/__main__.py
+++ b/Lib/asyncio/__main__.py
@@ -95,7 +95,7 @@ def run(self):
 
             console.write(banner)
 
-            if startup_path := os.getenv("PYTHONSTARTUP"):
+            if not sys.flags.isolated and (startup_path := os.getenv("PYTHONSTARTUP")):
                 sys.audit("cpython.run_startup", startup_path)
 
                 import tokenize
diff --git a/Lib/test/test_repl.py b/Lib/test/test_repl.py
index 042aa84b35dcf8..b4b2fc78e14091 100644
--- a/Lib/test/test_repl.py
+++ b/Lib/test/test_repl.py
@@ -28,7 +28,7 @@
     raise unittest.SkipTest("test module requires subprocess")
 
 
-def spawn_repl(*args, stdout=subprocess.PIPE, stderr=subprocess.STDOUT, custom=False, **kw):
+def spawn_repl(*args, stdout=subprocess.PIPE, stderr=subprocess.STDOUT, custom=False, isolated=True, **kw):
     """Run the Python REPL with the given arguments.
 
     kw is extra keyword args to pass to subprocess.Popen. Returns a Popen
@@ -42,7 +42,10 @@ def spawn_repl(*args, stdout=subprocess.PIPE, stderr=subprocess.STDOUT, custom=F
     # path may be used by PyConfig_Get("module_search_paths") to build the
     # default module search path.
     stdin_fname = os.path.join(os.path.dirname(sys.executable), "<stdin>")
-    cmd_line = [stdin_fname, '-I']
+    cmd_line = [stdin_fname]
+    # Isolated mode implies -EPs and ignores PYTHON* variables.
+    if isolated:
+        cmd_line.append('-I')
     # Don't re-run the built-in REPL from interactive mode
     # if we're testing a custom REPL (such as the asyncio REPL).
     if not custom:
@@ -215,7 +218,7 @@ def make_repl(env):
         with os_helper.temp_dir() as tmpdir:
             script = os.path.join(tmpdir, "pythonstartup.py")
             with open(script, "w") as f:
-                f.write("print('from pythonstartup')" + os.linesep)
+                f.write("print('from pythonstartup')\n")
 
             env = os.environ.copy()
             env['PYTHONSTARTUP'] = script
@@ -296,19 +299,27 @@ def test_asyncio_repl_reaches_python_startup_script(self):
         with os_helper.temp_dir() as tmpdir:
             script = os.path.join(tmpdir, "pythonstartup.py")
             with open(script, "w") as f:
-                f.write("print('pythonstartup done!')" + os.linesep)
-                f.write("exit(0)" + os.linesep)
+                f.write("print('pythonstartup done!')\n")
+            env = os.environ.copy()
+            env["PYTHON_HISTORY"] = os.path.join(tmpdir, ".asyncio_history")
+            env["PYTHONSTARTUP"] = script
+            p = spawn_asyncio_repl(isolated=False, env=env)
+            output = kill_python(p)
+            self.assertEqual(p.returncode, 0)
+            self.assertIn("pythonstartup done!", output)
 
+    def test_asyncio_repl_respects_isolated_mode(self):
+        with os_helper.temp_dir() as tmpdir:
+            script = os.path.join(tmpdir, "pythonstartup.py")
+            with open(script, "w") as f:
+                f.write("print('should not print')\n")
             env = os.environ.copy()
             env["PYTHON_HISTORY"] = os.path.join(tmpdir, ".asyncio_history")
             env["PYTHONSTARTUP"] = script
-            subprocess.check_call(
-                [sys.executable, "-m", "asyncio"],
-                stdout=subprocess.PIPE,
-                stderr=subprocess.PIPE,
-                env=env,
-                timeout=SHORT_TIMEOUT,
-            )
+            p = spawn_asyncio_repl(isolated=True, env=env)
+            output = kill_python(p)
+            self.assertEqual(p.returncode, 0)
+            self.assertNotIn("should not print", output)
 
     @unittest.skipUnless(pty, "requires pty")
     def test_asyncio_repl_is_ok(self):
diff --git a/Misc/NEWS.d/next/Library/2025-12-21-17-24-29.gh-issue-140648.i8dca6.rst b/Misc/NEWS.d/next/Library/2025-12-21-17-24-29.gh-issue-140648.i8dca6.rst
new file mode 100644
index 00000000000000..9e56f096b938f1
--- /dev/null
+++ b/Misc/NEWS.d/next/Library/2025-12-21-17-24-29.gh-issue-140648.i8dca6.rst
@@ -0,0 +1,3 @@
+The :mod:`asyncio` REPL now respects the :option:`-I` flag (isolated mode).
+Previously, it would load and execute :envvar:`PYTHONSTARTUP` even if the
+flag was set. Contributed by Bartosz Sławecki.