Background <p>Multiple sclerosis (MS) is a chronic neuroinflammatory and demyelinating condition that causes movement problems due to a probable failure to differentiate oligodendrocyte precursor cells into myelinating oligodendrocytes.</p> Aims <p>This work aims to evaluate the effect of exosomes derived from human umbilical cord mesenchymal stem cells on functional recovery in an animal model of multiple sclerosis.</p> Methods <p>The adult male C57BL/6J mice were randomly divided into three groups. The control mice were provided with a standard diet (CONT group). The chronically demyelinated (CPZ) group was fed a 0.2% cuprizone diet for 12 weeks. In the third group, mice received exosomes weekly for three weeks following chronic demyelination (Exo/CPZ group). The mice in the groups were tested weekly for mobility using a beam walking test (BWT), and their corpus callosum was studied histologically, immunohistochemically, and molecularly.</p> Results <p>According to BWT results, hucMSC-Exos enhanced motor function in mice following cuprizone intoxication. Histological staining revealed a substantial increase in remyelination in the corpus callosum. Moreover, immunohistochemical tests revealed that hucMSC-Exos lowered the level of demyelination and improved glial response by lowering the number of microglia and astrocytes. In addition, hucMSC-Exos also upregulated the expression of genes associated with the oligodendrocyte lineage.</p> Conclusions <p>hucMSC-Exos administration in the animal model of chronic MS leads to improved movement function, likely due to increased remyelination, modulation of inflammatory processes, and increased expression of genes related to the oligodendrocyte lineage. Therefore, using hucMSC-Exos seems to be a promising treatment strategy for demyelinating illnesses such as MS.</p> Graphical Abstract <p></p>