diff --git a/litellm/proxy/proxy_server.py b/litellm/proxy/proxy_server.py
index 33d84cd70789..34a8e5a89fd6 100644
--- a/litellm/proxy/proxy_server.py
+++ b/litellm/proxy/proxy_server.py
@@ -772,7 +772,13 @@ async def proxy_startup_event(app: FastAPI):  # noqa: PLR0915
         premium_user = _license_check.is_premium()
 
     ## CHECK MASTER KEY IN ENVIRONMENT ##
-    master_key = get_secret_str("LITELLM_MASTER_KEY")
+    # Only overwrite master_key from env var if it is actually set.
+    # initialize() may have already configured master_key from a config file;
+    # unconditionally assigning None here would discard that value when the
+    # LITELLM_MASTER_KEY env var is absent (e.g. programmatic startup, tests).
+    _env_master_key = get_secret_str("LITELLM_MASTER_KEY")
+    if _env_master_key is not None:
+        master_key = _env_master_key
     ### LOAD CONFIG ###
     worker_config: Optional[Union[str, dict]] = get_secret("WORKER_CONFIG")  # type: ignore
     env_config_yaml: Optional[str] = get_secret_str("CONFIG_FILE_PATH")
diff --git a/tests/test_litellm/proxy/test_preserve_master_key.py b/tests/test_litellm/proxy/test_preserve_master_key.py
new file mode 100644
index 000000000000..acb17c38f0d7
--- /dev/null
+++ b/tests/test_litellm/proxy/test_preserve_master_key.py
@@ -0,0 +1,49 @@
+"""
+Regression test for #22330:
+A master_key set by initialize() (via config file) must be preserved when
+LITELLM_MASTER_KEY is not set as an environment variable.
+"""
+
+from unittest.mock import AsyncMock, patch
+
+import pytest
+
+from litellm.proxy import proxy_server
+
+
+@pytest.mark.asyncio
+async def test_master_key_preserved_when_env_var_absent():
+    """proxy_startup_event must NOT overwrite a config-provided master_key."""
+    config_key = "sk-from-config-file-1234"
+
+    # Simulate initialize() having set master_key from a config file
+    original_master_key = proxy_server.master_key
+    proxy_server.master_key = config_key
+
+    try:
+        with patch(
+            "litellm.proxy.proxy_server.get_secret_str", return_value=None
+        ), patch(
+            "litellm.proxy.proxy_server.get_secret", return_value=None
+        ), patch(
+            "litellm.proxy.proxy_server.init_verbose_loggers"
+        ), patch(
+            "litellm.proxy.proxy_server._license_check"
+        ):
+            # proxy_startup_event is an @asynccontextmanager, so we must
+            # enter it with `async with` — a bare `await` would only create
+            # the generator object without executing the function body.
+            try:
+                async with proxy_server.proxy_startup_event(app=AsyncMock()):
+                    pass
+            except Exception:
+                # Startup will fail on DB/router setup — we only care about
+                # the master_key guard at the top of the function.
+                pass
+
+        assert proxy_server.master_key == config_key, (
+            f"master_key was overwritten: expected {config_key!r}, "
+            f"got {proxy_server.master_key!r}"
+        )
+    finally:
+        proxy_server.master_key = original_master_key