The mechanism by which oestrogens suppress experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, is only partially understood. We here demonstrate that treatment with 17β-oestradiol (E2) in C57BL/6 mice boosted the expression of programmed death 1 (PD-1), a negative regulator of immune responses, in the CD4+ FoxP3+ regulatory T (Treg) cell compartment in a dose-dependent manner that correlated with the efficiency of EAE protection. Administration of E2 at pregnancy levels but not lower concentrations also enhanced the frequency of Treg cells. Additionally, E2 treatment drastically reduced the production of interleukin-17 (IL-17) in the periphery of immunized mice. However, E2 treatment did not protect against EAE or suppress IL-17 production in PD-1 gene-deficient mice. Finally, E2 failed to prevent Treg-deficient mice from developing spontaneous EAE. Taken together, our results suggest that E2-induced protection against EAE is mediated by upregulation of PD-1 expression within the Treg-cell compartment.