Interleukin (IL)-17A and IL-17F are highly homologous, form heteroduplex, and bind the same receptor. However, their functional differences are still obscure. In this study, we found that the development of Dextran Sodium Sulfate (DSS)-induced colitis was ameliorated in Il17f–/–, but not Il17a–/–, mice. Upon transfer of CD25–CD45RBhiCD4+ T cells into Rag2–/– mice, Il17f−/− T cells induced significantly milder colitis in recipients than Il17a−/− T cells, accompanied with increased intestinal Foxp3+ regulatory T cells. Expanded-colonization of commensal Clostridium cluster XIVa (C. XIVa) was detected in both Il17f−/− mice and Il17f−/− T cell-transferred mice, associated with a decrease of two antimicrobial proteins , which preferentially restricted C. XIVa growth. IL-17F, but not IL-17A, is constitutively produced by naïve T cells and various types of colonic cells under physiological conditions. Furthermore, we found that treatment with anti-IL-17F, but not anti-17A antibody, suppressed the development of colitis. Targetting on intestinal IL-17F rather than IL-17A may provide a new strategy for treatment of inflammatory bowel disease.