The function of the myelinating system is important because a defective myelin sheath results in various nervous disorders, including multiple sclerosis and peripheral neuropathies. The dorsal root entry zone (DREZ) is a transitional area between the central nervous system (CNS) and the peripheral nervous system (PNS) that is generated by two types of cells-oligodendrocytes and Schwann cells (SCs). It is well known that after injury the extracellular matrix, including the CSPG, impairs axonal myelination by activating protein tyrosine phosphatase-sigma (PTP sigma) in both cells. The Intracellular Sigma Peptide (ISP) is memetic of the PTP sigma wedge region. It competitively binds to PTP sigma and regulates the downstream signaling of RhoA. In the present study, we aimed to investigate whether the ISP increased myelination in vivo and in vitro. The in vitro assay was meant to further verify the in vivo mechanisms. We observed that ISP administration could increase axonal myelination both in vivo and in vitro. Furthermore, we provide evidence that, in oligodendrocytes and Schwann cells, the myelination-induced effects of ISP application entail an inverse expression of the RhoA/CRMP2 signaling pathway. Overall, our results indicate that the ISP modulation of PTPs enhances axonal myelination via the RhoA/CRMP2 signaling pathways.