Prostaglandin E₂ (PGE₂) plays an important role in the regulation of duodenal bicarbonate (HCO₃^-) secretion, but its signaling pathway(s) are not fully understood. In the present study, we investigated the signaling pathways involved in PGE₂-mediated duodenal HCO₃^- secretion. Murine duodenal mucosal HCO₃^- secretion was examined in vitro in Ussing chambers by pH stat titration in the presence of a variety of signal transduction modulators. Phosphatidylinositol-3 kinase (PI3K) activity was measured by immunoprecipitation of PI3K and ELISA, AKT phosphorylation by Western Analysis with anti-Phospho-AKT and anti-AKT antibodies. PGE₂-stimulated duodenal HCO₃^- secretion was reduced by the cAMP-dependent signaling pathway inhibitors MDL-12330A and KT5720 by 23% and 20%, respectively, the Ca²⁺-influx inhibitor verapamil by 26%, the calmodulin antagonist W-13 by 24%, whereas the PI3K inhibitors wortmannin and LY294002 reduced PGE₂-stimulated HCO₃^- secretion by 51% and 47%, respectively. Neither mitogen-activated protein kinase (MAPK) inhibitor PD98059 nor tyrosine kinase inhibitor genistein altered PGE₂-stimulated HCO₃^- secretion. PGE₂ application caused a rapid and concentration-dependent increase in duodenal mucosal PI3K activity and AKT phosphorylation. These results demonstrated that PGE₂ activates PI3K in duodenal mucosa, and stimulates duodenal HCO₃^- secretion via cAMP-, Ca²⁺-, and PI3K-dependent signaling pathways.