Background: Radiotherapy is one of the most important factors which results in negative effects on wound healing and increases anastomosis leakage. Diverting loop ileostomy has been usually performed after colorectal anastomosis in cases of colorectal cancer with a history of neoadjuvant radiotherapy to decrease the chance of leakage. Considering the side effects of diverting loop ileostomy, the objective of the present study is to investigate the effect of human amniotic membrane (HAM) on colorectal anastomosis leakage after neo-adjuvant radiotherapy. Methods: In this experimental animal study, 20 crossbreed rabbits were randomly divided into two groups (case group: 13 rabbits, control group: 7 rabbits) after receiving an equal dose of external beam radiation. Four weeks after irradiation, resection of 4 cm of colorectal segment and end-to-end single layer anastomosis were conducted. In the case group, a 2×2 cm wrap of HAM applied around the site of anastomosis. Eight weeks later, all the survived rabbits were sacrificed. A segment of anastomotic sites was resected in all expired and survived rabbits and sent for pathological evaluation. Mann-Whitney U Test (SPSS for Windows, Ver. 16, Chicago, IL) was applied to analyze healing scores between the two groups.Results: Due to anastomosis dehiscence, 5 rabbits expired in the control group, but all the 13 rabbits (case group) survived after 8 weeks and showed no leakage. In addition, pathological evaluation revealed significant epithelialization and neovascularization in the case group. Statistically, healing score was higher in the case group rather than the control group (P<0.001).Conclusion: To prevent post irradiation colorectal anastomosis leakage, the use of HAM might play a significant role and a feasible technical approach.