It is now clear that functional CD4(+)CD25(+) regulatory T (T(R)) cells exist as part of the normal immune population and prevent the development of intestinal inflammation. We have recently shown that CD4(+)CD25(+) T(R) cells reside in the intestine and control intestinal homeostasis in humans and mice. In this study, we demonstrate that the TNF family molecule RANKL and its receptor RANK are critically involved in controlling the function of CD4(+)CD25(+) T(R) cells in the intestine. We first found that RANKL was preferentially expressed on both CD4(+)CD25(+) T(R) cells and colitogenic CD4(+) T cells, whereas RANK was expressed on dendritic cells. Although neutralizing anti-RANKL mAb did not affect T(R) activity of CD4(+)CD25(+) T(R) cells to suppress the proliferation of CD4(+) responder cells in vitro, in vivo administration of anti-RANKL mAb abrogated CD4(+)CD25(+) T(R) cell-mediated suppression of colitis induced by adoptive transfer of CD4(+)CD45RB(high) T cells into SCID mice. Interestingly, an adoptive transfer experiment using Ly5.1(+)CD4(+)CD45RB(high) cells and Ly5.2(+)CD4(+)CD25(+) T(R) cells revealed that the ratio of CD4(+)CD25(+) T(R) cells in total CD4(+) T cells in inflamed mucosa was significantly decreased by anti-RANKL mAb treatment. Consistent with this, the expression of RANK on lamina propria CD11c(+) cells from colitic mice was significantly increased as compared with that from normal mice, and in vitro treatment with anti-RANKL mAb suppressed the expansion of CD4(+)Foxp3(+) T(R) cells in culture with colitic lamina propria CD11c(+) cells. Together, these results suggest that the RANK-RANKL signaling pathway is critically involved in regulating the function of CD4(+)CD25(+) T(R) cells in colitis.