D-Myo-inositol (3,4,5,6) tetrakisphosphate [Ins(3,4,5,6)P₄] or phosphatidylinositol 3-kinase (PI 3-kinase) activity acts to inhibit calcium-dependent chloride secretion in T84 colonic epithelial cells. To further distinguish between the contributions of these two signaling pathways to the inhibition of secretion, we studied effects of insulin, because the insulin receptor links to PI 3-kinase but not to pathways postulated to generate Ins(3,4,5,6)P₄. Chloride secretion across T84 cell monolayers was studied in Ussing chambers. Activation of PI 3-kinase was assessed by Western blotting. Basolateral, but not apical, addition of insulin inhibited carbachol- and thapsigargin-induced chloride secretion in a time- and concentration-dependent fashion. Insulin-like growth factor-I (IGF-I) had similar effects. Insulin had no effect on Ins(3,4,5,6)P₄ levels, and the inhibitory effects of insulin and IGF-I on chloride secretion were fully reversed by the PI 3-kinase inhibitors wortmannin and LY-294002. Western blot analysis showed that both insulin and IGF-I recruited the 85-kDa regulatory and 110-kDa catalytic subunits of PI 3-kinase to anti-phosphotyrosine immunoprecipitates. In conclusion, insulin and IGF-I act to inhibit calcium-dependent chloride secretion through a PI 3-kinase-dependent pathway. Because insulin is released in a pulsatile fashion postprandially and IGF-I levels are elevated in pathological settings, our findings may have physiological and/or pathophysiological significance.