Bile acids are known to induce Ca²⁺ signals in pancreatic acinar cells. We recently showed that phosphatidylinositol 3-kinase (PI3K) regulates changes in free cytosolic Ca²⁺ ([Ca²⁺]i) elicited by cholecystokinin (CCK) by inhibiting sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA). The present study sought to determine whether PI3K regulates bile acid-induced [Ca²⁺]i responses. In pancreatic acinar cells, pharmacologic inhibition of PI3K with LY294002 or wortmannin inhibited [Ca²⁺]i responses to taurolithocholic acid 3-sulfate (TLC-S) and taurochenodeoxy-cholate (TCDC). Furthermore, genetic deletion of PI3K gamma isoform also decreased [Ca²⁺]i responses to bile acids. Depletion of CCK-sensitive intracellular Ca²⁺ pools or application of caffeine inhibited bile acid-induced [Ca²⁺]i signals, indicating that bile acids release Ca²⁺ from agonist-sensitive ER stores via IP₃-dependent mechanism. The PI3K inhibitors increased the amount of Ca²⁺ in intracellular stores during the exposure of acinar cells to bile acids, suggesting that PI3K negatively regulates the SERCA-dependent Ca²⁺ reloading into ER. Bile acids inhibited Ca²⁺ reloading into ER in permeabilized acinar cells. This effect was augmented by phosphatidylinositol 3,4,5-trisphosphate (PIP₃) suggesting that both bile acids and PI3K act synergistically to inhibit SERCA. Furthermore, inhibition of PI3K by LY294002 completely inhibited trypsinogen activation caused by TLC-S. Our results indicate that PI3K and its product, PIP₃, facilitate bile acid-induced [Ca²⁺]i responses in pancreatic acinar cells through inhibition of SERCA-dependent Ca²⁺ reloading into ER and that bile acid-induced trypsinogen activation is mediated by PI3K. The findings have important implications for the mechanism of acute pancreatitis since [Ca²⁺]i increases and trypsinogen activation mediate key pathologic processes in this disorder.