Inflammatory bowel disease is a serious health problem. Although it has been widely investigated, treatment of inflammatory bowel diseases currently remains as a challenging clinical problem. Over production of nitric oxide has been demonstrated to cause tissue damage and inflammation. In this study, the effect of methylene blue (MB), a well-known inhibitor of nitric oxide synthesis, was investigated in acetic acid (AA)-induced colitis model in Sprague-Dawley rats. Eighty male rats randomized into 4 groups (control, control MB, colitis, colitis + MB). AA was applied to groups 3 and 4. MB was added into group 2 and 4. Three days later, animals were sacrificed and 8 cm distal colonic segment resected and the specimens are examined using macroscopical, histological, and biochemical methods. The results of the macroscopic and microscopic examination showed that in group 4 the mucosal damage and inflammation score significantly lower than group 3. Increased intestinal permeability in acetic acid-administered group was significantly reversed by MB application. Myeloperoxidase activity and malondialdehyde levels increased significantly, while superoxide dismutase and catalase activities were suppressed after AA-administration. These biochemical parameters were reversed in MB-treated group. Administration of acetic acid resulted in increased levels of tumor necrosis factor-α, interleukin-1β, interleukin-6, total nitrite/nitrate levels and nitric oxide synthase activity. These biochemical alterations were significantly reversed by MB application also. In conclusion, our results indicate that MB decreases the level of nitric oxide and decreases inflammation in acetic acid-induced colitis.