diff --git a/homeassistant/components/cloudflare_r2/backup.py b/homeassistant/components/cloudflare_r2/backup.py
index d684542bd207..1279c1a2ba49 100644
--- a/homeassistant/components/cloudflare_r2/backup.py
+++ b/homeassistant/components/cloudflare_r2/backup.py
@@ -196,44 +196,46 @@ class R2BackupAgent(BackupAgent):
         )
         upload_id = multipart_upload["UploadId"]
         try:
-            parts = []
+            parts: list[dict[str, Any]] = []
             part_number = 1
-            buffer_size = 0  # bytes
-            buffer: list[bytes] = []
+            buffer = bytearray()  # bytes buffer to store the data
 
             stream = await open_stream()
             async for chunk in stream:
-                buffer_size += len(chunk)
-                buffer.append(chunk)
+                buffer.extend(chunk)
+
+                # upload parts of exactly MULTIPART_MIN_PART_SIZE_BYTES to ensure
+                # all non-trailing parts have the same size (required by S3/R2)
+                while len(buffer) >= MULTIPART_MIN_PART_SIZE_BYTES:
+                    part_data = bytes(buffer[:MULTIPART_MIN_PART_SIZE_BYTES])
+                    del buffer[:MULTIPART_MIN_PART_SIZE_BYTES]
 
-                # If buffer size meets minimum part size, upload it as a part
-                if buffer_size >= MULTIPART_MIN_PART_SIZE_BYTES:
                     _LOGGER.debug(
-                        "Uploading part number %d, size %d", part_number, buffer_size
+                        "Uploading part number %d, size %d",
+                        part_number,
+                        len(part_data),
                     )
                     part = await self._client.upload_part(
                         Bucket=self._bucket,
                         Key=self._with_prefix(tar_filename),
                         PartNumber=part_number,
                         UploadId=upload_id,
-                        Body=b"".join(buffer),
+                        Body=part_data,
                     )
                     parts.append({"PartNumber": part_number, "ETag": part["ETag"]})
                     part_number += 1
-                    buffer_size = 0
-                    buffer = []
 
             # Upload the final buffer as the last part (no minimum size requirement)
             if buffer:
                 _LOGGER.debug(
-                    "Uploading final part number %d, size %d", part_number, buffer_size
+                    "Uploading final part number %d, size %d", part_number, len(buffer)
                 )
                 part = await self._client.upload_part(
                     Bucket=self._bucket,
                     Key=self._with_prefix(tar_filename),
                     PartNumber=part_number,
                     UploadId=upload_id,
-                    Body=b"".join(buffer),
+                    Body=bytes(buffer),
                 )
                 parts.append({"PartNumber": part_number, "ETag": part["ETag"]})
 
diff --git a/tests/components/cloudflare_r2/test_backup.py b/tests/components/cloudflare_r2/test_backup.py
index 30b1637ffc02..c721468e80f3 100644
--- a/tests/components/cloudflare_r2/test_backup.py
+++ b/tests/components/cloudflare_r2/test_backup.py
@@ -367,6 +367,57 @@ async def test_agents_upload_network_failure(
     assert "Upload failed for cloudflare_r2" in caplog.text
 
 
+async def test_multipart_upload_consistent_part_sizes(
+    hass: HomeAssistant,
+    mock_client: MagicMock,
+    mock_config_entry: MockConfigEntry,
+) -> None:
+    """Test that multipart upload uses consistent part sizes.
+
+    S3/R2 requires all non-trailing parts to have the same size. This test
+    verifies that varying chunk sizes still result in consistent part sizes.
+    """
+    agent = R2BackupAgent(hass, mock_config_entry)
+
+    # simulate varying chunk data sizes
+    # total data: 12 + 12 + 10 + 12 + 5 = 51 MiB
+    chunk_sizes = [12, 12, 10, 12, 5]  # in units of 1 MiB
+    mib = 2**20
+
+    async def mock_stream():
+        for size in chunk_sizes:
+            yield b"x" * (size * mib)
+
+    async def open_stream():
+        return mock_stream()
+
+    # Record the sizes of each uploaded part
+    uploaded_part_sizes: list[int] = []
+
+    async def record_upload_part(**kwargs):
+        body = kwargs.get("Body", b"")
+        uploaded_part_sizes.append(len(body))
+        return {"ETag": f"etag-{len(uploaded_part_sizes)}"}
+
+    mock_client.upload_part.side_effect = record_upload_part
+
+    await agent._upload_multipart("test.tar", open_stream)
+
+    # Verify that all non-trailing parts have the same size
+    assert len(uploaded_part_sizes) >= 2, "Expected at least 2 parts"
+    non_trailing_parts = uploaded_part_sizes[:-1]
+    assert all(size == MULTIPART_MIN_PART_SIZE_BYTES for size in non_trailing_parts), (
+        f"All non-trailing parts should be {MULTIPART_MIN_PART_SIZE_BYTES} bytes, got {non_trailing_parts}"
+    )
+
+    # Verify the trailing part contains the remainder
+    total_data = sum(chunk_sizes) * mib
+    expected_trailing = total_data % MULTIPART_MIN_PART_SIZE_BYTES
+    if expected_trailing == 0:
+        expected_trailing = MULTIPART_MIN_PART_SIZE_BYTES
+    assert uploaded_part_sizes[-1] == expected_trailing
+
+
 async def test_agents_download(
     hass_client: ClientSessionGenerator,
     mock_client: MagicMock,