Demyelination disrupts neuronal function, making remyelination essential for recovery. Astrocytes play a dual role, either supporting or inhibiting this regeneration. This study investigates how IMT504, a 24-nucleotide non-CpG oligodeoxynucleotide with immunomodulatory properties, influences astrocyte function during remyelination, building on previous work (Mathieu et al., 2024) showing its benefits in neuroinflammation and oligodendrogenesis. In this work cuprizone (CPZ)-treated rats were subcutaneously injected IMT504 5 days before CPZ withdrawal and astrocytosis and astrogliosis were analyzed in corpus callosum and cerebral cortex 1h (T0), 3, 7 and 10 days after the last injection, during the remyelination process. Primary astrocyte cultures were also used to analyze IMT504 direct effects. Our results demonstrate that IMT504 regulates astrocyte function in vivo and in vitro. In vivo experiments showed that IMT504 modifies astrocyte´s GFAP expression and morphology in the corpus callosum and cortex of demyelinated animals. In astrocytes cultures, it inhibits proliferation, modulates phagocytosis, decreases migration, changes gene expression towards an anti-inflammatory phenotype, and acts on different signaling pathways. These findings suggest that IMT504 could improve CNS remyelination capacity by modulating astrocyte function, highlighting its therapeutic potential for demyelinating disorders like Multiple Sclerosis.