Intestinal regulatory T cells (T_reg cells) are necessary for the suppression of excessive immune responses to commensal bacteria. However, the molecular machinery that controls the homeostasis of intestinal T_reg cells has remained largely unknown. Here we report that colonization of germ-free mice with gut microbiota upregulated expression of the DNA-methylation adaptor Uhrf1 in T_reg cells. Mice with T cell–specific deficiency in Uhrf1 (Uhrf1^fl/flCd4-Cre mice) showed defective proliferation and functional maturation of colonic T_reg cells. Uhrf1 deficiency resulted in derepression of the gene (Cdkn1a) that encodes the cyclin-dependent kinase inhibitor p21 due to hypomethylation of its promoter region, which resulted in cell-cycle arrest of T_reg cells. As a consequence, Uhrf1^fl/flCd4-Cre mice spontaneously developed severe colitis. Thus, Uhrf1-dependent epigenetic silencing of Cdkn1a was required for the maintenance of gut immunological homeostasis. This mechanism enforces symbiotic host-microbe interactions without an inflammatory response.