Migration of olfactory ensheathing cells (OECs) is critical for development of olfactory system and essential for neural regeneration after OEC transplantation into nerve injury site. However, the molecular mechanisms underlying the regulation of directional migration of OECs remain unclear. In this study, we found that in migrating OECs, phosphorylated myosin light chain (p-MLC, active myosin II) displayed a polarized distribution, with the leading front exhibiting higher than soma and trailing process. Over-expression of GFP-MLC significantly reduced OEC migration. Moreover, decreasing this front-to-rear difference of myosin II activity by the frontal application of a ML-7 (myosin II inhibitors) gradient induced the collapse of leading front and reversed soma translocation of OECs, whereas, increasing this front-to-rear difference of myosin II activity by the rear application of a ML-7 or BDM gradient or the frontal application of a Caly (myosin II activator) gradient accelerated the soma translocation of OECs. Finally, myosin II as a downstream signaling of repulsive factor Slit-2 mediated the reversal of soma translocation induced by Slit-2. Taken together, these results suggest that the polarized distribution of active myosin II regulates the directional migration of OECs during spontaneous migration or upon to extracellular stimulation such as Slit-2.