BACKGROUND: Adipose-derived stem cells (ADSCs) exert immunomodulatory effects in the treatment of transplant rejection. This study aimed to evaluate the effects of ADSCs on the skin graft survival in a human-to-rat xenograft transplantation model and to compare single and multiple injections of ADSCs. METHODS: Full-thickness human skin xenografts were transplanted into the backs of Sprague–Dawley rats. The rats were injected subcutaneously on postoperative days 0, 3, and 5. The injections were as follows: triple injections of phosphate-buffered saline (PBS group), a single injection of ADSCs and double injections of PBS (ADSC 9 1 group), and triple injections of ADSCs (ADSC 9 3 group). The immunomodulatory effects of ADSCs on human skin xenografts were assessed. RESULTS: Triple injections of ADSCs considerably delayed cell-mediated xenograft rejection compared with the PBS and ADSC 9 1 groups. The vascularization and collagen type 1–3 ratios in the ADSC 9 3 group were significantly higher than those in the other groups. In addition, intragraft infiltration of CD3-, CD4-, CD8-, and CD68-positive cells was reduced in the ADSC 9 3 group. Furthermore, in the ADSC 9 3 group, the expression levels of proinflammatory cytokine interferon-gamma (IFN-c) were decreased and immunosuppressive prostaglandin E synthase (PGES) was increased in the xenograft and lymph node samples. CONCLUSION This study presented that triple injections of ADSCs appeared to be superior to a single injection in suppressing cell-mediated xenograft rejection. The immunomodulatory effects of ADSCs are associated with the downregulation of IFN-c and upregulation of PGES in skin xenografts and lymph nodes.