Multiple sclerosis (MS) is an autoimmune disorder characterized by demyelination and neurodegeneration in the central nervous system (CNS); severe symptoms lead MS patients to use complementary treatments. Ketogenic diet (KD) shows wide neuroprotective effects, but the precise mechanisms underlying the therapeutic activity of KD in MS are unclear. The present study established a continuous 24 days experimental autoimmune encephalomyelitis (EAE) mouse model with or without KD. The changes in motor function, pathological hallmarks of EAE, the status of microglia, neuroinflammatory response and intracellular signaling pathways in mice were detected by the rotarod test, histological analysis, real-time PCR (RT-PCR) and western blotting. Our results showed that KD could prevent motor deficiency, reduce clinical scores, inhibit demyelination, improve pathological lesions and suppress microglial activation in the spinal cord of EAE mice. Meanwhile, KD shifted microglial polarization toward the protective M2 phenotype and modified the inflammatory milieu by downregulating the production of pro-inflammatory cytokines, including TNF-α, IL-1β and IL-6, as well as upregulating the release of anti-inflammatory cytokines such as TGF-β. Furthermore, KD decreased the expression levels of CCL2, CCR2, CCL3, CCR1, CCR5, CXCL10 and CXCR3 in the spinal cord and spleen with reduced monocyte/macrophage infiltration in the CNS. In addition, KD inhibits NLRP3 activation in the microglia, as revealed by the significantly decreased co-expression of NLRP3+ and Iba-1+ in the KD + EAE group. Further studies demonstrated that KD suppresses inflammatory response and M1 microglial polarization by inhibiting the TLR4/MyD88/NF-κB/NLRP3 pathway, the JAK1/STAT1 pathway, HDAC3 and P2X7R activation, as well as up-regulation of JAK3/STAT6.