Identifying novel and safe immunosuppressants is of crucial importance. Recently, there have been several studies revealing that botulinum toxin A (BoTA) significantly alleviates ischemia-reperfusion injuries. Emerging evidence shows that ischemia-reperfusion injuries contribute to innate immune activation, promoting rejection, and inhibiting tolerance. Therefore, we hypothesized that a pretreatment with BoTA might decrease allograft rejection in a rat transplantation model. Twenty-four Lewis (LEW) rats were randomly assigned into two groups consisting of 12 rats each, depending on whether skin allograft was performed after pretreatment with BoTA (BoTA group) or with normal saline (control group). The experimental group was pretreated with a subcutaneous injection of BoTA (10 IU), while the control group was pretreated with normal saline 5 days prior to surgery. The donor Brown-Norway (BN) rat dorsal skin was subsequently grafted to the recipient LEW rats. The recipient wounds, measuring 2 cm × 2 cm, were made via dorsal skin excision through the panniculus carnosus. The donor skins of the same dimensions were obtained and transplanted on to the wounds and sutured with 4-0 nylon sutures. Mean graft survival time was measured in both groups. Quantitative reverse-transcriptase PCR and Western blotting were performed to evaluate the gene/protein expression of CD4 and VEGF. The mean graft survival time in the BoTA group was significantly longer than that of the control group (P=0.004). The relative mRNA and protein expression of CD4 was significantly lower in the BoTA group (P<0.001), while the relative mRNA and protein expression of VEGF was significantly higher in the BoTA group (P<0.001). In conclusion, our results show that BoTA prolongs the survival of skin allografts in a rat transplantation model.