Background: : Radiotherapy is an important factor which results in increase of anastomosis leakage. Diverting loop ileostomy has been usually performed after colorectal anastomosis with history of neo-adjuvant radiotherapy to decrease the chance of leakage. Considering this effect, we assessed the feasibility and outcome of human amniotic membrane in rectal anastomosis in dogs previously treated by high-dose radiotherapy. Materials and Methods: Twelve cross-breed male dogs with the age of 6-8 months and weighting 10-15 kg were randomly divided into four groups. Groups 1 and 2 received radiotherapy and 4 weeks later, single layer end to end anastomosis was performed for all the dogs; also, diverting loop ileostomy was placed in groups 1 and 3. Four weeks later, the anastomosis site was resected and sent for pathologic wound healing scoring. Data were analyzed by SPSS 16 software using Mann- Whitney test. Results: Two dogs (of group 1 and 2) died of peritonitis due to anastomosis leakage during the 2nd post operation week. There was no statistically significant difference in wound healing between the case and control groups (P-Value: 0.01). Conclusion: HAM had a protective role in colorectal anastomosis after neo-adjuvant radiation in cases without loop diverting ileostomy. It can be concluded that HAM placement is a feasible technique instead of diverting loop ileostomy in cases with neo-adjuvant radiotherapy and also in the benign colorectal diseases with high risk anastomoses.