Multiple sclerosis (MS) is one of the most common neurodegenerative diseases, causing demyelination and inflammation in the central nervous system. The pathology of MS has been extensively studied using the experimental autoimmune encephalomyelitis (EAE) mouse model. However, the molecular mechanisms are still largely unclear and require further investigation. In this study, we carried out quantitative proteomic analysis of the brain and spinal cord tissues in mice induced with EAE using a data-independent acquisition strategy and identified 744 differentially regulated proteins in the brain and 741 in the spinal cord. The changed proteins were highly related with phagocytosis, lysosomal enzymes, inflammasome activation, complements, and synaptic loss processes. Moreover, gene set enrichment analysis revealed the elevation of the SUMOylation process in EAE with the increase of SUMOylation-related enzymes and modification targets. Furthermore, to test the possibility of treating MS by targeting SUMOylation, we explored the application of a selective SUMO E1 inhibitor, TAK-981. Intriguingly, TAK-981 suppressed the global SUMOylation level in the brain and significantly alleviated the symptoms of EAE in mice. Our findings contribute to a better understanding of MS pathology, reveal the important role of SUMOylation in disease progression, and demonstrate the potential of the SUMO E1 inhibitor as a novel treatment for MS.