Aim: One of the most troublesome complications of anal preserving surgery is anal sphincter dysfunction. The aim of this study was to evaluate functional recovery after implantation of adipose-derived stem cell (ADSC) sheets, novel biotechnology, for an anal sphincter resection animal model. Methods: Eighteen female Sprague-Dawley rats underwent removal of the nearest half of the internal and external anal sphincter muscle. Nine rats received transplantation with ADSC sheets to the resected area while the remaining rats received no transplantation. The rats were evaluated for the anal function by measuring their resting pressure before surgery and on postoperative days 1, 7, 14, 28, and 56. In addition, the rats were examined for the presence of smooth muscle and also to determine its origin. Results: The improvement of the anal pressure was significantly greater in the ADSC sheet transplantation group compared with the control group. Histologically, at the vicinity of the remaining smooth muscle, reproduction of smooth muscle was detected. Using in fluorescence in situ hybridization, the cells were shown to be from the recipient. Conclusion: Regenerative therapy using ADSC sheet has the potential to recover anal sphincter dysfunction due to anorectal surgery.