Neurons have been classically considered insulin-insensitive cells. In this in vitro study, the effect of insulin on neuronal glucose uptake was studied by assaying glucose uptake, translocation of glucose transporter isoform GLUT3, and fusion of GLUT3 vesicles with the plasma membrane. Insulin (50 nM) promoted translocation of GLUT3 to the plasma membrane. However, insulin neither promoted fusion of GLUT3 with the plasma membrane nor increased neuronal glucose uptake. In cells pre-exposed to insulin, depolarization with 40 mM KCl markedly increased fusion of GLUT3 with plasma membrane and neuronal uptake of glucose. Based on these data, we propose that insulin regulates neuronal glucose uptake by promoting translocation of GLUT3 to the plasma membrane, and that insulin enables neurons to respond to demand for energy induced by increased neuronal activity.