Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis, characterized by neuroinflammation, demyelination, and astrocyte activation. In this study, we investigated the therapeutic potential of intrathecal (IT) administration of umbilical cord-mesenchymal stem cells (UC-MSCs). UC-MSCs represent a promising candidate for therapeutic applications owing to their immunomodulatory properties and minimal immunogenicity. In a MOG35-55-induced EAE model, UC-MSC treatment reduced clinical scores and body weight loss, indicating suppression of disease progression. On day 21, UC-MSCs did not significantly affect mRNA levels of inflammatory cytokines and chemokines in the spleen or spinal cord, nor did they modulate B- and T-cell functions. Interestingly, UC-MSCs reduced the mRNA expression levels of astrocyte activation markers such as GFAP and LCN2 in the spinal cord. Conversely, UC-MSCs markedly upregulated the mRNA expression levels of neurotrophic factors BDNF and GDNF compared to the control group. Histological analysis showed that UC-MSCs reduced demyelination and suppressed GFAP and IBA-1 activation. Collectively, UC-MSCs may represent a promising therapeutic strategy for demyelinating diseases by alleviating EAE symptoms through astrocyte modulation and upregulation of neurotrophic factors.