Dietary compounds as well as commensal microbiota contribute to the generation of a unique gut environment. In this study, we report that dietary folic acid (FA) is required for the maintenance of Foxp3+ regulatory T cells (Tregs) in the colon. Deficiency of FA in the diet resulted in marked reduction of Foxp3+ Tregs selectively in the colon. Blockade of folate receptor 4 and treatment with methotrexate, which inhibits folate metabolic pathways, decreased colonic Foxp3+ Tregs. Compared with splenic Tregs, colonic Tregs were more activated to proliferate vigorously and were highly sensitive to apoptosis. In colonic Tregs derived from mice fed with a FA-deficient diet, expression of anti-apoptotic molecules Bcl-2 and Bcl-xL was severely decreased. A general reduction of peripheral Tregs was induced by a neutralizing Ab against IL-2, but a further decrease by additional FA deficiency was observed exclusively in the colon. Mice fed with an FA-deficient diet exhibited higher susceptibility to intestinal inflammation. These findings reveal the previously unappreciated role of dietary FA in promotion of survival of Foxp3+ Tregs that are in a highly activated state in the colon.