Demyelination is defined as the loss of the myelin sheath surrounding axons, while remyelination restores this sheath and contributes to functional recovery. Multiple sclerosis is a common inflammatory demyelinating disorder in which the remyelination process is frequently unsuccessful. IMT504 is a synthetic 24-base non-CpG oligodeoxynucleotide containing two characteristic PyNTTTTGT motifs. Considering the regenerative and immunomodulatory properties of IMT504, and given that our group first reported its promyelinating benefits with effects on neuroinflammation and remyelination in an animal model, the present study aims to investigate the effects of IMT504 both in vitro, in glial cell cultures, and in vivo, in the corpus callosum (CC) of cuprizone (CPZ)-demyelinated rats. Our findings show that IMT504: (i) enhances microglial cell phagocytosis, (ii) induces the activation of p38, ERK1/2, SAPK/JNK, and NF-kB signaling in microglial cells, (iii) induces the activation of p38 and ERK1/2 signaling in oligodendrocyte precursor cells, and (iv) produces changes in the activation of MAPK signaling in the CC of CPZ-demyelinated rats. These results underscore the prospective therapeutic value of IMT504, reinforcing its potential role in the development of interventions for demyelinating disorders.