A progressive decline in the integrity of the immune system is one of the physiologic changes during aging. The frequency of autoimmune diseases or immune disorders increases in the aging population, but the state of regulatory T (Treg) cells in aged individuals has not been well determined. In the present study, we investigated the levels, phenotypes, and function of CD4⁺CD25⁺ Treg cells in Balb/c mice, which were older than 20 months. Significantly enhanced percentages of CD4⁺CD25⁺ Treg cells in the periphery (blood, spleen, and lymph nodes) of the aged mice were observed. These Treg cells showed modified Vβ family distribution, reduced levels of CD45 receptor B and CD62 ligand molecules, as well as normal levels of forkhead box p3. However, when the inhibiting function of Treg cells was assayed in the in vitro assays and in a delayed-type hypersensitivity (DTH) model, CD4⁺CD25⁺ Treg cells of aged mice displayed significantly lower inhibiting ability on alloantigen-induced DTH reaction or cytokine productions (IL-2 and IFN-γ) but not cell proliferation of effector T cells, as compared with CD4⁺CD25⁺ Treg cells of young mice. In addition, the percentages of CD4⁺CD8^–CD25⁺ Treg cells in the thymi of aged mice increased significantly, but their total cell numbers decreased markedly in these mice. Our present studies indicated collectively that the percentages, phenotypes, the size of TCR repertoire, and function of CD4⁺CD25⁺ Treg cells were altered significantly with aging in mice. The functional defects of CD4⁺CD25⁺ Treg cells may shed light on the role of CD4⁺CD25⁺ Treg cells in the increased sensitivity to autoimmune diseases of aged populations.