Gut commensal microbes shape the mucosal immune system by regulating the differentiation and expansion of several types of T cell(1-5). Clostridia, a dominant class of commensal microbe, can induce colonic regulatory T (T-reg) cells, which have a central role in the suppression of inflammatory and allergic responses(3). However, the molecular mechanisms by which commensal microbes induce colonic T-reg cells have been unclear. Here we show that a large bowel microbial fermentation product, butyrate, induces the differentiation of colonic T-reg cells in mice. A comparative NMR-based metabolome analysis suggests that the luminal concentrations of short-chain fatty acids positively correlates with the number of T-reg cells in the colon. Among short-chain fatty acids, butyrate induced the differentiation of T-reg cells in vitro and in vivo, and ameliorated the development of colitis induced by adoptive transfer of CD4(+) CD45RB(hi) T cells in Rag1(-/-) mice. Treatment of naive T cells under the T-reg-cell-polarizing conditions with butyrate enhanced histone H3 acetylation in the promoter and conserved non-coding sequence regions of the Foxp3 locus, suggesting a possible mechanism for how microbial-derived butyrate regulates the differentiation of T-reg cells. Our findings provide new insight into the mechanisms by which host-microbe interactions establish immunological homeostasis in the gut.