Ulcerative colitis is a chronic recurrent inflammatory bowel disease in which oxidative stress has been implicated. The aim of the present study was to evaluate possible protective effects of N-acetylcysteine against acetic acid-induced colitis in a rat model. Rats were administered intrarectal saline (control group) or acetic acid (colitis model group). Rats with acetic acid-induced colitis were treated by intraperitoneal or intrarectal administration of N-acetylcysteine (500 mg/kg) (treated group). Another series of rats were pre-treated by intraperitoneal or intrarectal administration of N-acetylcysteine, then administered intrarectal acetic acid (pre-treated group). The degree of tissue injuries was assessed by macroscopical and histopathological scores of the colonic mucosa. Malondialdehyde, myeloperoxidase, reduced glutathione, superoxide dismutase, and catalase levels were measured in tissue extracts of the dissected colon. Administration of N-acetylcysteine intraperitoneally or intrarectally ameliorated macroscopic score alterations produced by acetic acid in treated groups. In addition, microscopical improvement was observed in all N-acetylcysteine-treated rats compared to untreated animals with colitis. In the colonic tissues of the acetic acid-induced colitis, myeloperoxidase activity and malondialdehyde levels were elevated, while the reduced glutathione levels and superoxide dismutase and catalase activities were decreased. However, intraperitoneal or intrarectal treatment with N-acetylcysteine reversed these parameters, compared to the untreated colitis group. Notably, intrarectal administration of N-acetylcysteine elevated the reduced glutathione levels more markedly compared to the other treatment groups. Superoxide dismutase levels were increased in intraperitoneally or intrarectally N-acetylcysteine-treated groups significantly compared to the control, colitis and pre-treated groups. But there was no significant increase in catalase activity. In conclusion, N-acetylcysteine could be beneficial as a complementary agent in treatment of ulcerative colitis.