Colonic administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) induced acute colitis in mice and elicited a Th1 immune response. Th17 cells are believed to play a major role in TNBS-induced colitis. The aim of this study is to investigate the roles of interleukin (IL)-17 and interferon (IFN)-γ in the pathogenesis of TNBS-induced acute colitis. We assessed the inflammation scores of TNBS-induced acute colitis in wild-type (WT), IL-17 knockout (KO), and IFN-γ KO mice and measured the levels of inflammatory cytokines using real-time PCR and ELISAs. Histology data showed that IL-17 KO mice with TNBS-induced colitis had significantly lower neutrophil infiltration and inflammatory macroscopic scores compared to the IFN-γ KO mice and WT mice. Intraperitoneal injection of anti-IL-17 monoclonal antibody confirmed a specific role for IL-17 in TNBS-induced acute colitis in the 3 strains of mice. The severity of colitis was higher in IFN-γ KO mice and lower in IL-17 KO mice compared to WT mice. Our data suggested that IL-17 signaling plays a critical role in the local inflammation of TNBS-induced colitis, while IFN-γ was not an important mediator of the local inflammation response. IL-17 may represent a target for therapeutic intervention in inflammatory bowel disease patients.