G protein-coupled receptor 3 (GPR3) is highly expressed in various neurons, and unique in its ability to constitutively activate the Gαs protein without the addition of ligands, which elevates the basal level of intracellular cAMP. In our earlier study, we clarified that GPR3 play a role in both neurite outgrowth and neuronal survival in developing cerebellar granular neurons. However, the potential role of GPR3 in the axon regeneration still remains unclear. Herein, we investigated the possible involvement of GPR3 in axonal regeneration after optic nerve injury in mice. GPR3 expression was observed in the retinal ganglion cells (RGCs). GPR3 expression was increasingly expressed during development in the mouse primary retinal ganglion neurons (RGNs). When GPR3 expression was suppressed by siRNA, both neurite outgrowth and survival in RGNs were significantly inhibited. In the pressure-induced retinal ischemia model, the number of RGCs and the inner plexiform layer thickness were significantly reduced in the GPR3 knockout mice compared to those in wild-type mice 7 days after the induction of transient retinal ischemia. In addition, regenerating axon was significantly increased 4 weeks after optic nerve crush (ONC) when GPR3 was overexpressed in RGCs using adeno-associated virus. Furthermore, axonal regeneration was dramatically augmented when Zymosan was inoculated in the GPR3-overexpressed retina. These results suggest that GPR3 play a potential role in accelerating axonal regeneration after ONC in mouse.