The migration of olfactory ensheathing cells (OECs) is essential for pioneering the olfactory nerve pathway during development and for promoting axonal regeneration when implanted into the injured central nervous system (CNS). In the present study, recombinant Nogo-66 enhanced the adhesion of OECs and inhibited their migration. Using immunocytochemistry and western blot, we showed that the Nogo-66 receptor (NgR) was expressed on OECs. When NgR was released from the cell surface with phosphatidylinositol-specific phospholipase C or neutralized by NgR antibody, the effect of Nogo-66 on OEC adhesion and migration was markedly attenuated. Nogo-66 was found to promote the formation of focal adhesion in OECs and inhibited their membrane protrusion through the activation of RhoA. Furthermore, the co-culture migration assay demonstrated that OEC motility was significantly restricted by Nogo-A expressed on Cos7 cell membranes or oligodendrocytes. Moreover, treatment with anti-NgR antibody facilitated migration of implanted OECs in a spinal cord hemisection injury model. Taken together, we demonstrate, for the first time, that Nogo, a myelin-associated inhibitor of axon regeneration in the CNS, enhances the adhesion and inhibits the migration of OECs via NgR regulation of RhoA.