Abstract: This study systematically investigated the therapeutic mechanism of matrine derivative, (6aS, 10S, 11aR, 11bR, 11cS)-10-methylaminododecahydro-3a, 7a-diaza-benzo (de) anthracene-8-thione (MASM), in experimental autoimmune encephalomyelitis (EAE) through multi-dimensional analysis. The myelin oligodendrocyte glycoprotein MOG_35-55/pertussis toxin-induced EAE mouse model was established, and neurological impairment was confirmed using Kono's 5-point scoring system, mice with scores between 0.5 and 3.5 were considered successful models. These mice were randomly divided into six groups: EAE control, MASM groups (1.5, 3 and 6 mg·kg^-1), a positive control group (fingolimod, 1.0 mg·kg^-1), and a normal control group. Drug administration was initiated on the day of grouping, once daily for 21 consecutive days. The Kono's score was used to evaluate the neurological function; HE staining and electron microscopy was used to observe the pathological changes of spinal cord; transforming growth factor-β1 and monocyte chemoattractant protein-1 content was detected by ELISA. The gene expression of H2T23 was detected by qRT-PCR, the binding interactions between MASM and human frizzled homolog (FZD5) was evaluated by molecular docking, and protein expression levels of glial fibrillary acidic protein (GFAP), complement 3 (C3), wingless type mouse mammary tumor virus integration site family member 5a (Wnt5a), FZD5, protein kinase Cα (PKCα), and the phosphorylation level of glycogen synthase kinase 3 (GSK3β) were detected by Western blot. MASM significantly improved neurological function, reduced spinal cord inflammation and demyelination, and downregulated astrocyte activation markers (GFAP, C3, H2T23) (P < 0.05, P < 0.01) and Wnt5a/FZD5 pathway components (Wnt5a, FZD5, PKCα, p-GSK3β) (P < 0.05, P < 0.01). MASM demonstrates strong binding capacity to FZD. These findings suggest that MASM alleviates EAE by inhibiting astrocyte polarization via the Wnt5a/FZD5 pathway. The animal study was approved by the Animal Ethics Committee of Fujian University of Traditional Chinese Medicine (approval number: FJTCMIACUC 2023319).