CD4+ CD25+ regulatory T cells in normal animals are engaged in the maintenance of immunological self-tolerance and prevention of autoimmune disease. However, accumulating evidence suggests that a fraction of the peripheral CD4+ CD25− T cell population also possesses regulatory activity in vivo. Recently, it has been shown glucocorticoid-induced TNFR family-related gene (GITR) is predominantly expressed on CD4+ CD25+ regulatory T cells. In this study, we show evidence that CD4+ GITR+ T cells, regardless of the CD25 expression, regulate the mucosal immune responses and intestinal inflammation. SCID mice restored with the CD4+ GITR− T cell population developed wasting disease and severe chronic colitis. Cotransfer of CD4+ GITR+ population prevented the development of CD4+ CD45RB high T cell-transferred colitis. Administration of anti-GITR mAb-induced chronic colitis in mice restored both CD45RB high and CD45RB low CD4+ T cells. Interestingly, both CD4+ CD25+ and CD4+ CD25− GITR+ T cells prevented wasting disease and colitis. Furthermore, in vitro studies revealed that CD4+ CD25− GITR+ T cells as well as CD4+ CD25+ GITR+ T cells expressed CTLA-4 intracellularly, showed anergic, suppressed T cell proliferation, and produced IL-10 and TGF-β. These data suggest that GITR can be used as a specific marker for regulatory T cells controlling mucosal inflammation and also as a target for treatment of inflammatory bowel disease.