diff --git a/.changelog/5320.fixed b/.changelog/5320.fixed
new file mode 100644
index 00000000000..9df4767330c
--- /dev/null
+++ b/.changelog/5320.fixed
@@ -0,0 +1 @@
+`opentelemetry-instrumentation-urllib3`: keep the pool default headers when `urlopen` is called without headers
diff --git a/instrumentation/opentelemetry-instrumentation-urllib3/src/opentelemetry/instrumentation/urllib3/__init__.py b/instrumentation/opentelemetry-instrumentation-urllib3/src/opentelemetry/instrumentation/urllib3/__init__.py
index 18a149e369b..8651791ee05 100644
--- a/instrumentation/opentelemetry-instrumentation-urllib3/src/opentelemetry/instrumentation/urllib3/__init__.py
+++ b/instrumentation/opentelemetry-instrumentation-urllib3/src/opentelemetry/instrumentation/urllib3/__init__.py
@@ -442,7 +442,7 @@ def instrumented_urlopen(wrapped, instance, args, kwargs):
             return wrapped(*args, **kwargs)
 
         # avoid modifying original headers on inject
-        headers = headers.copy() if headers is not None else {}
+        headers = headers.copy() if headers is not None else instance.headers.copy()
 
         sanitized_method = sanitize_method(method)
         span_name = _get_span_name(sanitized_method)
diff --git a/instrumentation/opentelemetry-instrumentation-urllib3/tests/test_urllib3_integration.py b/instrumentation/opentelemetry-instrumentation-urllib3/tests/test_urllib3_integration.py
index 4916c2ec71e..3f05bada92d 100644
--- a/instrumentation/opentelemetry-instrumentation-urllib3/tests/test_urllib3_integration.py
+++ b/instrumentation/opentelemetry-instrumentation-urllib3/tests/test_urllib3_integration.py
@@ -960,3 +960,16 @@ def test_urlopen_mixed_args(self):
         self.assertEqual(b"Hello!", response.data)
         span = self.assert_span()
         self.assertEqual(span.attributes["http.request.header.x_test"], ("Value",))
+
+    def test_urlopen_pool_headers(self):
+        URLLib3Instrumentor().uninstrument()
+        URLLib3Instrumentor().instrument(captured_request_headers=["X-Test"])
+        pool = urllib3.HTTPConnectionPool("mock", headers={"X-Test": "Value"})
+        response = pool.urlopen("GET", "/status/200")
+        self.assertEqual(b"Hello!", response.data)
+        span = self.assert_span()
+        headers = dict(Mocket.last_request().headers)
+        self.assertEqual(headers.get("x-test"), "Value")
+        self.assertIn("traceparent", headers)
+        self.assertEqual(span.attributes["http.request.header.x_test"], ("Value",))
+        self.assertEqual(pool.headers, {"X-Test": "Value"})