Dimethyl fumarate (DMF) is an oral agent for relapsing-remitting multiple sclerosis (RRMS). In this study, we investigated the therapeutic mechanism of DMF using experimental autoimmune encephalomyelitis (EAE). DMF treatment decreased the proliferation of T cells and the production of IL-17A and GM-CSF. DMF treatment also decreased the development and/or infiltration of macrophages in the central nervous system (CNS), and reduced the ratio of iNOS⁺ pro-inflammatory macrophage versus Ym1⁺ immunomodulatory macrophages. Furthermore, DMF treatment suppressed the deposition of complement C3 (C3) and development of reactive C3⁺ astrocytes. The decrease in iNOS⁺ macrophages, C3⁺astrocytes, and C3 deposition in the CNS resulted in the reduction in demyelination and axonal loss. This study suggests that the beneficial effects of DMF involve the suppression of iNOS⁺ pro-inflammatory macrophages, C3⁺ astrocytes, and deposition of C3 in the CNS.