A 9-year-old Holstein cow was evaluated for colic and decreased milk production of 2 days' duration. Preoperative serum biochemical results suggested hepatic damage and cholestasis. On the basis of persistent signs of abdominal pain that were nonresponsive to analgesics, exploratory laparotomy was performed. The cow was found to have choleliths. Cholecystocentesis was performed, and samples were submitted for cytologic examination and bacterial culture. Bacterial culture yielded Escherichia coli and Clostridium perfringens. Using digital pressure, choleliths were reduced until they could be passed through the bile duct into the duodenum. The cow recovered from surgery without complications, and all serum biochemical test results returned to reference ranges. Cholelithiasis is rare in cattle but can result in signs of abdominal pain.