Although phosphatidylinositol 3-kinase (PI3K) is essential for several cellular signal transductions, its role in the regulation of cystic fibrosis transmembrane conductance regulator (CFTR) activity in intestinal epithelial cells is poorly understood. Therefore, the possible involvement of PI3K in the regulation of cAMP- and cGMP-induced duodenal epithelial CFTR activation was investigated in the present study. Forskolin and 8-Br-cGMP markedly stimulated duodenal mucosal HCO₃^- secretion and short-circuit current (I_sc) in CFTR wild type mice, which was significantly inhibited by CFTR_inh-172, a highly potent and specific CFTR inhibitor. Forskolin and 8-Br-cGMP failed to stimulate duodenal HCO₃^- secretion and Isc in CFTR knockout mice. Moreover, forskolin- and 8-Br-cGMP-stimulated duodenal HCO₃^- secretion and Isc were significantly reduced by wortmannin and LY294002, two selective PI3K inhibitors that are structurally and mechanistically different. Forskolin and 8-Br-cGMP induced CFTR phosphorylation and shifted CFTR proteins to the plasma membrane of duodenal epithelial cells, which were inhibited by wortmannin and LY294002. Forskolin and 8-Br-cGMP not only increased the activity of PI3K, but also induced the phosphorylation of Akt, a signaling molecule downstream of PI3K, which were again inhibited by wortmannin and LY294002. Taken together, our results obtained from functional, biochemical and morphological studies demonstrated that PI3K pathway plays an important role in the regulation of cAMP- and cGMP-induced duodenal epithelial CFTR channel activity and intracellular trafficking.