We investigated the molecular mechanism of neurite outgrowth with PACAP treatments using a rat adrenal-derived pheochromocytoma cell line-PC12. This study was specifically investigated into the regulation of PACAP-induced CRMP2 previously identified in a mouse brain ischemia model and which could be recovered by PACAP treatment. We have previously revealed that PACAP-mediated neuroprotection involves not only CRMP2 but also pathways related to GSK-3β and other signaling components. To clarify whether CRMP2 acts directly on PACAP or through GSK-3β as part of the mechanism of PACAP-induced neurite outgrowth, we observed neurite outgrowth in the presence of GSK-3β inhibitors and activators. Post PACAP treatment in PC12 cells, immunostaining was used to confirm protrusion elongation, while RT-PCR, 2D Gel with Western blotting, and inhibition experiments were performed to confirm the expression of the PACAP gene, its receptors, and downstream signaling components. These results indicate that neurite protrusion elongation by PACAP follows a GSK-3β-regulated pathway through the PAC1-R, where the AKT and cAMP/ERK pathways are involved. These findings also provide a solid basis for future research to develop new therapies and therapeutic agents to treat neural disorders and may contribute to neurogenesis.