The G protein-coupled receptor 3 (GPR3), a member of constitutively active Gαs protein receptor family, has potential for enhancing neurite outgrowth and neuronal survival. Recently, we have clarified that GPR3 is transported along the neurite, predominantly distributed at the tip of neurite, and thus contributed to the local protein kinase A (PKA) activation. In the present study, we aimed to determine the possible involvement of GPR3 in presynaptic function using PC12 cells. GPR3 mRNA was highly upregulated 12–48 h after nerve growth factor- and serum deprivation-induced differentiation. Likewise, synapsin 2 mRNA was increasingly upregulated until 48 h after stimulation. In PC12 cells transfected with a green fluorescent protein (GFP)-tagged GPR3 expressing vector, the fluorescence of GPR3-GFP was transported along the neurites and concentrated in the presynaptic terminals. Moreover, when the expression of GPR3 was suppressed by shRNA, the phosphorylation of synapsin decreased 28 h after stimulation, as shown by the Western blotting analysis. To further explore the roles of GPR3 in presynaptic function, we tested whether expression of GPR3 modulates the uptake and release of dopamine in neuronal cells. GPR3-knockdown PC12 cells showed an increased uptake of dopamine, whereas dopamine release was decreased upon phorbol ester-induced potentiation. These results suggest that GPR3 may serve as a potential modulator of presynaptic function.