During the course of neuronal development, an axon is specified by the PI3 kinase- and PKA-dependent signaling pathways. However, the up-stream factors that modulate the activity of these kinases remain to be fully elucidated. Recently, we clarified that the subcellular dynamics of Gαs activating G protein-coupled receptor 3 (GPR3) are associated with the local activation of PKA in cerebellar granular neurons. In the current study, we focused on the possible involvement of GPR3 in axon specification. The numbers of neurons with Tau-1-positive neurites significantly decreased at 48–60 h after GPR3 siRNA transfection in rat hippocampal neurons. In contrast, the upregulated expression of GPR3 resulted in an accelerated formation of Tau-1-positive neurites at 24 h after transfection. Similarly, a delayed formation of Tau-1-positive neurites was observed in the hippocampal neurons from GPR3-knockout mice. GPR3-mediated acceleration of axon formation was significantly reduced by the administration of PI3-kinase inhibitor and not by PKA inhibitor. Subsequently, we evaluated whether the GPR3 expression affects the de-phosphorylation of CRMP2, which is downstream in the PI3-kinase signaling pathway. The number of neurons with pCRMP2-negative neurites significantly decreased 60 h after GPR3 siRNA transfection. Furthermore, the staining intensity of pCRMP2 at the neurite tip was significantly stronger in GPR3-knockout mice than in the wild-type mice. Our cumulative results indicate the potential role of GPR3 in the axon specification in cultured hippocampal neurons.