Although certain treatment options exist for intestinal incontinence, none are curative. Adipose-derived stem cells (ADSCs) have emerged as promising therapeutic agents, but most preclinical studies of their effectiveness for anal function have used autologous or allogeneic ADSCs. In this study, the effectiveness, timing of administration, and required dosage of human ADSCs were investigated for clinical application. A 10-mm balloon catheter was used to induce anal sphincter injury in immunodeficient mice in the following experimental groups (n=4 per group): ADSC (injected ADSCs after injury), PBS (injected phosphate-buffered saline after injury), and control (uninjured). The effects of different timing (immediately after injection and 30 days following injury) and number of human ADSCs administered was compared among groups based on defecation status and pathological evaluation. In terms of defecation status, groups receiving ≥1×104 human ADSCs after injection showed improvement. Pathological images showed that compared to the PBS group, the thinnest part of the sphincter was thicker for animals that received ≥1×104 human ADSCs, and fibrosis of the sphincter was notable in those treated with 1×103 human ADSCs or PBS. Furthermore, defecation status was improved by administration of human ADSCs, not only immediately after injury, but also at 30 days following injury. Human ADSC administration in a mouse model of anal sphincter injury was effective. Injection of ≥1×104 human ADSCs was the amount necessary to improve defecation status, an effect detected in both the acute and chronic phases.