Protein kinase C (PKC) activation induces cellular reprogramming and differentiation in a variety of cellular models. Although many effectors of PKC physiological actions have been elucidated, the molecular mechanisms that regulate oligodendrocytes differentiation after PKC activation are still unclear. Here we applied a liquid chromatography - mass spectrometry (LC-MS/MS) approach to identify proteins, and biological processes, modulated after PKC activation in the established PKC-induced differentiation model, MO3.13 oligodendroglial cell line. Our analysis revealed that PKC signaling activation leads to the down-regulation of biological processes related to cell cycle and DNA replication and the up-regulation of a glial like developmental program. Our data suggested a role for ROCK in the modulation of MO3.13 phenotype, cytoskeletal and mechanotransduction dynamics but not in the modulation of cell cycle and calcium transport. Overall, our findings provides a resource for elucidating the PKC-mediated signaling network contributing to a more robust knowledge of the molecular dynamics leading to this cell fate transition.