Tributyrin (TBT) is a TAG composed of three butyric acids that has beneficial effects on ulcerative colitis due to its trophic, anti-inflammatory, pro-apoptotic and anti-carcinogenic properties. The goal of the present study was to evaluate the efficacy and mechanisms of action of TBT supplementation in the prevention of mucosal damage in experimental colitis. Mice received either a control diet or a TBT-supplemented diet for 15 d. Colitis was induced by dextran sodium sulphate administration during the last 7 d. Mucosal damage and the activation of immune cells and cytokines were determined by histological score, flow cytometry and ELISA. Leucocyte rolling and adhesion were assessed by intravital microscopy. Oxidative stress was determined by monitoring hydroperoxide concentration and evaluating superoxide dismutase (SOD) and catalase activities. Intestinal permeability was analysed using diethylenetriaminepentaacetate acid (99mTcDTPA). Compared with the colitis group, the animals in the colitis+TBT group had reduced mucosal damage and neutrophil and eosinophil mucosal infiltration, which were associated with a higher percentage of regulatory T cells (Treg) and higher levels of transforming growth factor β and IL-10 in the lamina propria. The level of in vivo leucocyte adhesion in the colon microvasculature was reduced after TBT supplementation. A lower level of hydroperoxide and higher levels of SOD and catalase activities were associated with TBT supplementation. TBT-supplemented mice showed reduced intestinal permeability to the levels intermediate between the control and colitis groups. In conclusion, the present results show that TBT has positive effects on colonic restructuring in experimental colitis. Additionally, TBT supplementation changes the immune response by controlling inflammation and regulating the expression of anti-inflammatory cytokines and Treg.