Naturally arising CD4⁺CD25⁺ regulatory T (T_R) cells have been shown to prevent and cure murine T cell-mediated colitis. However, their exact mechanism to control colitogenic memory CD4⁺ T cells in in vivo system excluding the initial process of naive T cell activation and differentiation has not been examined. Using the colitogenic effector-memory (T_EM) CD4⁺ cell-mediated colitis model induced by adoptive transfer of colitogenic CD4⁺CD44^highCD62L^- lamina propria (LP) T cells obtained from colitic CD4⁺CD45RB^high T cell-transferred mice, we here show that CD4⁺CD25⁺ T_R cells are able to not only suppress the development of colitis, Th1 cytokine productions, and the expansion of colitogenic LP CD4⁺ T_EM cells, but also extensively expand by themselves in vivo. In vitro co-culture assay revealed that CD4⁺CD25⁺ T_R cells proliferate in the presence of IL-2-producing colitogenic LP CD4⁺ T_EM cells at the early time point (48hr after culture), followed by the acquisition of suppressive activity at the late (96 hr after culture). Collectively, these data suggest the distinct timing of the IL-2-dependent expansion of CD4⁺CD25⁺ T_R cells and the their suppressive activity to colitogenic LP CD4⁺ T_EM cells.