The aim of this study was to evaluate the effects of melatonin on the inflammatory response and hydroxyproline production in an experimental acute and chronic model of trinitrobenzene sulfonic (TNBS) acid-induced colitis in Wistar rats. In the acute model, melatonin (0.5, 1, and 2 mg/kg, i.p.) was applied 48, 24, and 1 hr prior to the induction of colitis and 24 and 48 hr after; the severity of colitis was less evident in melatonin-treated animals with significant response in the group treated with 2 mg/kg. All doses investigated significantly reduced the myeloperoxidase activity (MPO). In the chronic studies, melatonin (1 and 2 mg/kg, i.p.) was administered daily 24 hr before hapten instillation and for 7 or 21 days after TNBS; melatonin (2 mg/kg) worsened colitis evolution in the 21-day study with a significant increase in MPO activity and tumor necrosis factor-alpha production with respect to TNBS group. Histological slides were in concordance with macroscopic data where areas of extensive necrosis and edema, fibrosis, and absence of regenerated epithelium were observed. Moreover, the hydroxyproline determination, used as indicator of collagen production and fibrosis, also showed a marker increase. The results obtained in this experimental model showed that short-term administration is protective while in the long term it negatively influences evolution of inflammatory colitis; therefore, the immunostimulatory effect of melatonin in some situations when given chronically, such as during inflammatory bowel disease, might lead to negative consequences.