Multiple sclerosis (MS) is a chronic autoimmune neurological disease characterized by inflammatory demyelination damage. Therapeutic alternatives for MS are still limited. Interventions to improve cholesterol homeostasis may be a viable approach to promoting the remyelination of MS patients. Analgecine (AGC), the extracts of Vaccinia-inoculated rabbit skin, is used in clinical for the treatment of low back pain. We previously found AGC had neuroprotective effect. In the present study, we aimed to discover the effect and mechanism of AGC on cholesterol homeostasis in a model of MS. In this study, we found that AGC effectively reduced the neurological deficit score and reversed the state of reduced body weight in the experimental autoimmune encephalomyelitis (EAE) mice. Furthermore, we also demonstrated that AGC treatment attenuated demyelination in the spinal cord and corpus callosum, amended loss of neurons in the hippocampal and cortical regions in EAE model. In addition, we identified the potential of AGC to well regulate cholesterol metabolism homeostasis by proteomic analysis, which may be related to its regulation of DHCR24 gene expression. Meanwhile, we also demonstrated in vitro and in vivo that AGC could effectively alleviate the abnormal cholesterol aggregation in microglia. Taken together, our study indicates that AGC is a promising potential agent for the treatment of MS by improving demyelination and neuron loss, which may be related to its ability in the regulation of cholesterol metabolism homeostasis.