Objectives: Adhesion formation continues to be an important problem in gastrointestinal surgery. In recent years, methylene blue (MB) has been reported to be an effective agent for preventing peritoneal adhesions. However, its effects on the wound healing process are unknown. In the present study, we investigated the effects of MB on the early and late phases of anastomotic wound healing and on adhesion formation. Methods: We randomly categorized 92 rats into 2 groups in bursting pressure measurements and 50 rats into 3 groups in the adhesion model. We divided the animals into saline-treated (n = 46) or MB-treated (n = 46) groups. Bursting pressures of the anastomoses were measured on postoperative days 3 and 7. In biochemical studies, tissue hydroxyproline levels, total nitrite/nitrate levels and nitric oxide synthase activity were measured on postoperative days 3 and 7. In the adhesion model, we randomly categorized rats into sham (n = 10), saline-treated (n = 20) and MB-treated (n = 20) groups, and the formation of intraperitoneal adhesions was scored on postoperative day 14. We compared the measurement of bursting pressure and biochemical measurements of tissue hydroxyproline levels, total nitrite/nitrate levels and nitric oxide synthase activity. Histopathological findings of specimens were presented. Results: During the early phase of wound healing (postoperative day 3), bursting pressures, tissue hydroxyproline, total nitrite/nitrate levels and nitric oxide synthase activity in the MB-treated group were significantly lower than those of the saline-treated group. On postoperative day 7, there was no significant difference in these parameters between MB and saline-treated groups. In the adhesion model, MB caused a significant reduction in the formation of peritoneal adhesions. Conclusion: MB prevents peritoneal adhesions but causes a significant impairment of anastomotic bursting pressure during the early phase of the wound healing process by its transient inhibitory effect on the nitric oxide pathway.