diff --git a/botocore/httpchecksum.py b/botocore/httpchecksum.py
index a4b7e8648d..5d0a6c17eb 100644
--- a/botocore/httpchecksum.py
+++ b/botocore/httpchecksum.py
@@ -409,6 +409,12 @@ def _apply_request_trailer_checksum(request):
         # services such as S3 may require the decoded content length
         headers["X-Amz-Decoded-Content-Length"] = str(content_length)
 
+        if "Content-Length" in headers:
+            del headers["Content-Length"]
+            logger.debug(
+                "Removing the Content-Length header since 'chunked' is specified for Transfer-Encoding."
+            )
+
     if isinstance(body, (bytes, bytearray)):
         body = io.BytesIO(body)
 
diff --git a/tests/functional/test_s3.py b/tests/functional/test_s3.py
index 1206b83272..0cee2ee726 100644
--- a/tests/functional/test_s3.py
+++ b/tests/functional/test_s3.py
@@ -1536,6 +1536,26 @@ def test_trailing_checksum_set(self):
         body = self.http_stubber.requests[0].body.read()
         self.assertIn(b"x-amz-checksum-crc32:eCQEmA==", body)
 
+    def test_trailing_checksum_set_with_content_length_removes_header(self):
+        with self.http_stubber:
+            self.client.put_object(
+                Bucket="foo", Key="bar", Body="baz", ContentLength=123
+            )
+        sent_headers = self.get_sent_headers()
+        self.assertEqual(sent_headers["Content-Encoding"], b"aws-chunked")
+        self.assertEqual(sent_headers["Transfer-Encoding"], b"chunked")
+        self.assertEqual(
+            sent_headers["X-Amz-Trailer"], b"x-amz-checksum-crc32"
+        )
+        self.assertEqual(sent_headers["X-Amz-Decoded-Content-Length"], b"3")
+        self.assertEqual(
+            sent_headers["x-amz-content-sha256"],
+            b"STREAMING-UNSIGNED-PAYLOAD-TRAILER",
+        )
+        body = self.http_stubber.requests[0].body.read()
+        self.assertIn(b"x-amz-checksum-crc32:eCQEmA==", body)
+        self.assertNotIn("Content-Length", sent_headers)
+
     def test_trailing_checksum_set_empty_body(self):
         with self.http_stubber:
             self.client.put_object(Bucket="foo", Key="bar", Body="")
@@ -1686,7 +1706,11 @@ def test_content_sha256_set_s3_on_outpost(self):
 class TestCanSendIntegerHeaders(BaseSessionTest):
     def test_int_values_with_sigv4(self):
         s3 = self.session.create_client(
-            "s3", config=Config(signature_version="s3v4")
+            "s3",
+            config=Config(
+                signature_version="s3v4",
+                request_checksum_calculation="when_required",
+            ),
         )
         with ClientHTTPStubber(s3) as http_stubber:
             http_stubber.add_response()