Background: Anastomotic leakage is a major factor for morbidity in colorectal surgery. Anastomotic reinforcement with biological or synthetic materials has been claimed to be useful in preventing anastomotic leakage. Methods: We evaluated a non-cross-linked collagenous matrix Bio-Gide® (BG) for sealing colonic anastomoses in a rodent model. The animals were investigated for 4, 30 and 90 days. Macroscopic examination, histological examination and measurement of bursting pressure were performed. The anastomotic stricture rate was evaluated by radiographic contrast enema. Results: Microscopically anastomoses sealed by BG showed impaired anastomotic healing. Blood vessel ingrowth and collagen deposition were decreased without reaching significance after 4 days. The anastomotic bursting pressure was significantly decreased (p = 0.0454) in the early phase of healing. Anastomotic neovascularization was significantly decreased compared to the control group after 30 (p = 0.0058) and 90 days (p = 0.0275). Although no difference in anastomotic stricture rate was evident, the rate of intra-abdominal adhesions was significantly increased after 30 (p = 0.0124) and 90 days (p = 0.0281). Conclusion: BG failed to improve colonic anastomotic healing. Early anastomotic healing was impaired if anastomoses were reinforced with BG. BG did not affect the anastomotic stricture rate for up to 3 months; nevertheless, intra-abdominal adhesions were increased.