Mechanisms maintaining the growth of a "semi-foreign" fetus within the maternal uterus via immune tolerance remain unclear. CD4(+)CD25(+) regulatory T (Treg) cells have been implicated in the maintenance of maternal-fetal immune tolerance. Additionally, 17β-estradiol (E2) is able to initiate immune suppression through CD4(+)CD25(+) Treg cells during early pregnancy. Little is known, however, regarding the relationship between progesterone (P4) and immune tolerance during midterm pregnancy, an important period, characterized by higher levels of P4 but lower levels of E2 in the serum. Here, we examined the effects of P4 on the expansion and function of systemic and local uterine CD4(+)CD25(+) Treg cells during midterm pregnancy in mice. Using in vivo and in vitro models, we provide the first evidence that P4 not only increases the proportion of CD4(+)CD25(+) Treg cells and IL-10 expression but also enhances their suppressive function. Moreover, at physiological doses relevant to midterm pregnancy, P4, but not E2, converts CD4(+)CD25(-) T cells into CD4(+)CD25(+) Treg cells. This conversion was inhibited in vitro by the nuclear P4 receptors antagonist RU 486 and in vivo in P4-treated ovariectomized and pseudopregnant mice models, suggesting that P4 expands Treg populations via nuclear P4 receptors. Furthermore, RU 486 significantly reduced the quantity and function of Treg cells in the fetal-maternal interface before the onset of induced abortion. Interestingly, with decreasing Foxp3, proinflammatory factors increased. Together, the present results demonstrate that P4 is an important regulator of systemic and local CD4(+)CD25(+) Treg cells, which are involved in maintaining maternal-fetal immune tolerance during midterm pregnancy.