Previous studies have demonstrated that vitamin D3-mediated protection in EAE occurs only in females and is dependent on the presence of diestrus levels of 17-estradiol (E2). To evaluate the role of estrogen receptors in vitamin D3 treatment of EAE, we compared disease severity, CNS histopathology and immunological responses in vehicle and calcitrol (1,25 dihydroxyvitamin D) treated WT C57BL/6 mice vs. GPR30 membrane estrogen receptor (MER) knockout mice with MOG-35-55 peptide-induced EAE. Our results demonstrated that vitamin D-mediated prevention of clinical signs, CNS cellular lesions and demyelination observed in WT mice was abrogated in GPR30-KO mice with EAE. Regulatory effects of vitamin D treatment that were MER dependent included increased levels of IL-10 and IL-6 secreted by MOG peptide-reactive splenocytes and increased expression of CCL5, CCR1 & CCR3 in spleen tissue. These results demonstrate for the first time that the MER is a key contributor to the E2-dependent effects of vitamin D-mediated protection in EAE.