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Background Secure closure of the colonic access site is one of the most important issues for the development of natural orifice transluminal endoscopic surgery. Objective To evaluate the feasibility, reproducibility, and efficacy of a new over-the-scope clip. Design Descriptive study, in vivo porcine colon. Intervention In 10 female Yorkshire pigs weighing 30 to 35 kg, a 10-mm colotomy was performed by using a needle-knife and advancing the endoscope to the peritoneal cavity. Colonic closure was performed by using the Padlock-G clip (Aponos Medical, Kingston, NH) delivered with the Lock-It system (Aponos). Main Outcome Measurements Animals were monitored daily for signs of peritonitis and sepsis over a period of 14 days. During necropsy, the peritoneal cavity was examined, and the colon segment containing the incision was excised for pathological study. Results Closure was achieved in all cases. Nine of 10 pigs survived 14 days without complications, but 1 pig was killed immediately after the procedure because of severe bleeding during the colonic incision. The median closure time was 8 minutes (range 1-30 minutes). At necropsy, adhesions were observed in 5 cases. The incision was hardly visible at the serosa side in 3 cases and was not identified in 6 cases. In 6 cases, the clip was still slightly attached to the mucosa, and it was not found in 3 cases. The pathological study revealed a complete remodeling of the colonic wall in all cases. Limitations Animal model, noncomparative study. Conclusion The Padlock-G clip procedure is feasible, reproducible, effective, and easy to perform, and it provides a reliable colonic closure.