Background: Perianal fistula is a complicated disorder and most difficult to manage. New treatment methods would help surgeons to achieve a better outcome in patients with perianal fistula. Human amniotic membrane (HAM) has positive effects on wound healing in several conditions. The present study aimed to further determine the effect of HAM on wound healing of perianal fistula in rabbits.Methods: In a prospective experimental study, 14 male rabbits (aged 4-6 months and weighing 3-4 kg) were randomly divided into 2 groups. After 12 weeks, the high type perianal fistula was repaired with endorectal flap (ERF) and ERF plus HAM in the control and case groups, respectively. In all rabbits of the case group, a 1×1 cm width wrap of HAM was applied and fixed around the ERF site. Three weeks later, the repaired site of the perianal fistula was sent for pathologic wound healing scoring. The results were analyzed with the SPSS 21.0 software using Mann-Whitney test. Results: Six rabbits of each group survived the study period. There was a statistically significant difference in wound healing between the case and control groups (P<0.001). Wound healing process in the case group occurred better and faster than the control group.Conclusion: HAM has an effective role in enhancing the ERF procedure and considered appropriate. A combination of HAM with other methods is recommended.