Clinical improvement during pregnancy in multiple sclerosis (MS) patients suggests that sex hormones exert potent regulatory effects on autoimmune function. Our previous studies demonstrated that estrogen- (17-estradiol; E2) mediated protection against experimental autoimmune encephalomyelitis (EAE), a mouse model for MS, hinges on the B cells, leading to elevated numbers of IL-10 secreting CD1d(hi)CD5(+) B regulatory cells (Bregs) in wild type mice. Our data show that co-administration of E2 and IL-10(+) B cells ameliorates EAE disease severity and limits CNS infiltrating leukocytes in B cell deficient mice. Additionally, treatment with E2 and Bregs reduces demyelination and dramatically decreases the proportion of CD11b(+)CD45(hi) activated microglia/macrophages found in the CNS of immunized animals compared to vehicle, E2 or Breg cells alone. Furthermore, mice given E2 and Bregs exhibit increased numbers of peripheral programmed death-1 positive CD4(+)Foxp3(+) regulatory T cells (Tregs) and up-regulation of programmed death receptor-ligand-1 and CD80 expression on monocytes. Our study suggests IL-10 producing Bregs have powerful therapeutic potential as an agent against EAE when augmented with E2 treatment.