A captive 19-yr-old female Pacific gopher snake (Pituophis catenifer) presented for evaluation after keeper staff noticed a midbody swelling. Physical examination revealed a 6.6 × 3 × 3-cm slightly mobile mass approximately midway between the head and tail. Distension of the gallbladder was diagnosed using radiographs, ultrasonography, and fine needle aspiration. Pre-operative coelomic ultrasound revealed close association between the gallbladder, aorta, and vena cava. Repeated cholecystocentesis failed to resolve the distension and a cholecystectomy was performed. Mycobacterium chelonae was isolated from the removed gallbladder and the snake was treated with azithromycin 10 mg/kg PO every 7 days for 6 months. Acid fast organisms were identified on a fecal smear during the peri-operative period. None of the usual clinical signs of mycobacteriosis in reptiles, such as respiratory disease, weight loss, or lethargy, were observed in the present case and the patient continues to thrive. Cholecystectomy is rarely reported in snakes, and this case describes an appropriate pre-operative evaluation, surgical approach, and postoperative management plan. This is the first report of antemortem diagnosis of cholecystitis in a snake caused by M. chelonae infection, which is a potentially zoonotic pathogen.