Objective The effects of seromuscular layer anastomosis， extramucosal anastomosis，single-layer anastomosis and double-layer anastomosis of gastrointestinal tract on anastomotic healing were compared. Methods Chinese rabbits were divided into four groups: group A (double-layer anastomosis, n=10), group B (single-layer inverted anastomosis, n=10), group C (extramucosal anastomosis, n=10) and group D (seromuscular layer anastomosis, n=10). Five anastomoses were performed in each animal: one side-to-side gastroduodenal anastomosis, two end-to-end ileal and colonic anastomoses respectively. Half of each group was sacrificed on postoperative day 3 and 7 respectively to determine in situ anastomostic bursting pressures (ABP) and hydroxyproline (HP) content, and to receive histopathologic examination. Inflammatory index and mucosal healing index of anastomosis were calculated. Results There were no significant differences in case of ABP among the groups on day 3, and with the same result among group A, B and C on day 7 in gastroduodenal, ileoileal and colocolonic anastomoses. On day 7, the ABP of gastroduodenal anastomosis was dramatically higher in group D than group A and B (P＜0.05), the ABP of ileoileal anastomosis in group D was significantly increased compared with group A (P＜0.01), and the ABP of colocolonic anastomosis in group D was also higher than group A, B and C (P＜0.05). There was no statistical difference in HP content among the 4 groups in gastroduodenal and ileal anastomoses on day 3 (Pgt;0.05), and in ileal and colonic anastomoses on day 7 (Pgt;0.05). HP content was higher in group A than group B on day 3 in colonic anastomoses (P＜0.05), and it was also found to be higher in group D than group A on day 7 in gastroduodenal anastomosis (P＜0.025). Inflammatory reaction was not different among the 4 groups in gastroduodenal and ileoileal anastomoses on day 3, and the inflammatory indices of gastroduodenal and colocolonic anastomoses in all groups were similar on day 7. The inflammatory index of colocolonic anastomosis was signicantly increased in group A than group C on day 3 (P＜0.05), and that of ileoileal anastomosis in group A was higher than group D on day 7 (P＜0.05). The mucosal healing indices of anastomoses were not significantly different among the 4 groups on day 7. Conclusion Seromuscular layer anastomosis of gastrointestinal tract is as safe as other hand-sewn anastomoses, but it is more convenient and simpler than others.