Autoimmune diseases including multiple sclerosis predominantly affect females. Although high levels of sex hormones, particularly estrogen (E2), can reduce proinflammatory immune responses, it remains unclear if a lack of endogenous sex hormones might affect treatment with exogenous sex hormones. Pretreatment with E2 almost completely prevents intact female and male mice from developing clinical and histological signs of experimental autoimmune encephalomyelitis (EAE) by promoting various regulatory immune cell phenotypes. To evaluate the effects of exogenous estrogen in the absence of endogenous sex hormones, the current study compared EAE severity and the emergence of different immunoregulatory cell populations after E2 pretreatment of ovariectomized (OVX) female versus male mice. We found that E2 equally protected both OVX females and males from EAE over a 21 day observation period concomitant with reduced total cell numbers in spleen and spinal cord (males only), but enhanced percentages of CD19(+)CD5(+)CD1d(hi), CD19(+)CD138(+)CD44(hi) and CD19(+)Tim-1(+) Breg cells, CD8(+)CD122(+) Treg cells and CD11b(+)CD 206(+)ARG-1(+) anti-inflammatory M2-like monocytes/macrophages in both groups. In contrast, E2 decreased the percentage of CD4(+)CD25(+)FoxP3(+) Treg cells in OVX females but increased these Treg cells in males and intact female mice. These data suggest that with the exception of CD4(+)CD25(+)FoxP3(+) Treg cells, E2 protection against EAE promotes highly overlapping immunoregulatory subsets in OVX females and males.