Si-Ni-San, a traditional Chinese medicinal formula, exerts an important function in the treatment of inflammatory bowel diseases based upon thousands of years of clinical practice, but the underlying mechanism is still unclear. In this study, we investigated the therapeutic potential of Si-Ni-San and its ingredient glycyrrhizin in trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis in mice, a well-characterized murine model for Crohn's disease. Si-Ni-San and glycyrrhizin significantly ameliorated TNBS-induced colitis with reduced mortality and recovery of body weights. In addition, Si-Ni-San and glycyrrhizin dose-dependently decreased macroscopic inflammation scores, microscopic histological scores, and myeloperoxidase activity. Furthermore, Si-Ni-San and glycyrrhizin caused a decrease in pro-inflammatory cytokines including IFN-gamma, IL-12, TNF-alpha and IL-17 and an increase in regulatory cytokine IL-10 in colon of the mice. It should be noticed the therapeutic effect of Si-Ni-San at 450 mg/kg was much better than that of its contained content of glycyrrhizin at 10 mg/kg. In conclusion, Si-Ni-San and glycyrrhizin significantly ameliorated TNBS-induced colitis in mice through regulating pro- and anti-inflammatory cytokine production.