The main obstacle to remyelination in demyelinating diseases, such as multiple sclerosis, is the inability of oligodendrocyte precursor cells (OPCs) to differentiate into mature oligodendrocytes (OLs) in the demyelinating region. Consequently, promoting OL differentiation and myelin remodeling is a key goal in the search for treatments. Rho GTPases play diverse and important roles throughout the development of neuronal axons and the formation of the myelin sheath. The current study aimed to investigate the direct protective effects of catalpol on demyelination damage induced by myelin oligodendrocyte glycoprotein (MOG) immunization and to explore whether the GEF-Cdc42/Rac1 signaling pathway contributes to the regeneration effect induced by catalpol. In the MOG-induced experimental autoimmune encephalomyelitis (EAE) mouse model of demyelination, we observed that catalpol significantly promoted OL development by enhancing the expression of glutathione S-transferase pi (GST-pi) in the affected brain. By Luxol fast blue staining and myelin basic protein (MBP) expression assessment, catalpol was found to increase MBP expression and promote myelin repair. Furthermore, catalpol promoted OL differentiation associated with the upregulation of Cdc42/Rac1 expression and activation in vivo. In addition, PAK1/MRCKα, proteins downstream of Cdc42/Rac1, was positively regulated by catalpol. We also found that catalpol alleviated clinical neurological dysfunction, inhibited inflammatory infiltration, increased the proportion of Treg cells, and suppressed demyelination. Overall, our study is the first to reveal that catalpol can promote OL generation and myelination and contributes to the crucial regulatory process of GEF-Cdc42/Rac1 signaling expression and activation. Therefore, catalpol is a promising drug candidate for the potential treatment of demyelinating diseases.