OBJECTIVES: To describe an open, beating heart surgical technique and use of a bovine pericardial prosthetic valve for mitral valve replacement (MVR) in the dog. STUDY DESIGN: Clinical case report. ANIMALS: Male Bull Terrier (17-month-old, 26 kg) with mitral valve dysplasia and severe regurgitation. METHODS: A bovine pericardial bioprosthesis was used to replace the mitral valve using an open beating heart surgical technique and cardiopulmonary bypass. RESULTS: Successful MVR was achieved using a beating heart technique. Mitral regurgitation resolved and cardiac performances improved (left ventricular end-diastolic diameter decreased from 57.6 to 48.7 mm, and left atrium/aorta ratio returned to almost normal, from 1.62 to 1.19). Cardiopulmonary by-pass time and total surgical duration were decreased compared with standard cardioplegic techniques. Surgical recovery was uneventful and on echocardiography 6 months later valve function was excellent. CONCLUSION: Considering the technique advantages (no cardiac arrest, ischemic reperfusion injury, and hypothermia, or the need for aortic dissection and cannulation for administration of cardioplegic solution), short-term mortality and morbidity may be reduced compared with standard cardioplegic techniques. CLINICAL RELEVANCE: Based on experience in this dog, beating heart mitral valvular replacement is a seemingly safe and viable option for the dog and bovine pericardial prosthesis may provide better long-term survival than mechanical prostheses.