The cytokines IL-17A and IL-17F have 50% amino-acid identity and bind the same receptor; however, their functional differences have remained obscure. Here we found that Il17f^–/– mice resisted chemically induced colitis, but Il17a^–/– mice did not, and that Il17f^−/− CD45RB^hiCD4⁺ T cells induced milder colitis in lymphocyte-deficient Rag2^–/– mice, accompanied by an increase in intestinal regulatory T cells (T_reg cells). Clostridium cluster XIVa in colonic microbiota capable of inducing T_reg cells was increased in both Il17f^−/− mice and mice given transfer Il17f^−/− T cells, due to decreased expression of a group of antimicrobial proteins. There was substantial production of IL-17F, but not of IL-17A, not only by naive T cells but also by various colon-resident cells under physiological conditions. Furthermore, antibody to IL-17F suppressed the development of colitis, but antibody to IL-17A did not. These observations suggest that IL-17F is an effective target for the treatment of colitis.