Multiple lines of evidence support the existence of crosstalk between the insulin receptor and G protein-coupled receptor (GPCR) signaling systems. However, the precise molecular mechanism(s) mediating this interaction is poorly understood. The results presented in this study show that exposure of ductal pancreatic adenocarcinoma BxPc-3, HPAF-II and PANC-1 cells to insulin for as little as 1 min rapidly enhanced the magnitude and the rate of increase in intracellular Ca²⁺ concentration produced by the GPCR agonists bradykinin, angiotensin II, vasopressin, neurotensin and bombesin. The potentiating effect of insulin was dose-dependent and it was produced in response to Gq protein-coupled, but not Gi protein-coupled, receptor agonists. Real-time imaging of single cells showed that treatment with insulin enhances the rate and magnitude of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] hydrolysis and generation of Ins(1,4,5)P₃ in response to GPCR stimulation. Short-term treatment with rapamycin, an mTOR inhibitor, completely abrogated the ability of insulin to increase the rate and magnitude of Ca²⁺ signaling and production of Ins(1,4,5)P₃ in response to bradykinin stimulation, indicating that insulin potentiates Gq protein-coupled receptor signaling through an mTOR-dependent pathway. We propose that the potentiation of GPCR signaling by insulin provides a mechanism by which insulin enhances cellular responsiveness to Gq protein-coupled receptor agonists, including GPCR-mediated autocrine and paracrine loops in cancer cells.