The activation of microglia is an important cause of central nervous system (CNS) inflammatory cell infiltration and inflammatory demyelination in multiple sclerosis (MS). NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome-mediated signaling plays a decisive role in microglial activation. Mitophagy is closely related to NLRP3-mediated neuroinflammation. Previous studies have shown that ketogenic diet (KD) suppresses microglial NLRP3 inflammasome activation and exerts mitophagy-stimulating effects, but the specific mechanism remains unclear. The current study examined the mechanism underlying the anti-inflammatory effect of KD on experimental autoimmune encephalomyelitis (EAE). Our data show that KD inhibited demyelination, increased co-staining of the translocase of the outer mitochondrial membrane (TOM20) and microtubule-associated protein 1A/1B-light chain 3 (LC3II), and decreased microglial NLRP3 inflammasome activation and histone deacetylase 3 (HDAC3) in the hippocampus of EAE mice. Further correlation analysis showed that the reduction of HDAC3 was negatively correlated with NLRP3 activation and positively correlated with the induction of mitophagy in KD-fed EAE mice. In BV2 microglial cells, we confirmed that the inhibition of HDAC3 promoted 5' adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/unc-51-like autophagy activating kinase (ULK)1 and PTEN-induced putative kinase 1 (PINK1)/Parkin-meditated mitophagy, which led to the up-regulation of acetylated AMPK, acetylated ULK1 and acetylated Parkin, and subsequently reduced ROS accumulation and inhibited the activation of the NLRP3 inflammasome. In addition, treatment with 3-methyladenine (3-MA), a specific autophagy inhibitor, abolished the anti-inflammatory effect of HDAC3 inhibition in BV2 cells. The study illustrates that KD ameliorates EAE by reducing NLRP3-mediated inflammation in microglial cells via HDAC3 inhibition and enhancement of mitophagy-related protein acetylation.