Olfactory ensheathing cells (OECs) are a unique type of macroglia with axonal growth-promoting properties. The migrating ability of OECs in CNS is essential for neural regeneration. However, little is known about the extracellular and intracellular factors that regulate OEC migration. In the present study, we examined the effects of glial cell line-derived neurotrophic factor (GDNF) on OECs migration. Initially, the "scratch" migration assay, Boyden chamber assay, and explant migration assay showed that GDNF could promote OECs migration in vitro. Treatment of OECs with GDNF also induced cytoskeleton reorganization and up-regulated expression of cytoskeleton proteins. GDNF-induced OECs migration was demonstrated depending on GFRalpha-1 and Ret receptor, and activation of JNK and Src signaling cascades. Furthermore, GDNF was found to promote implanted OECs migration in a spinal cord hemisection injury model. Together, we report, to our knowledge for the first time, that GDNF stimulate OECs migration in vitro and in vivo.