Several inositol-containing compounds play key roles in receptor-mediated cell signaling events. Here, we describe a function for a specific inositol polyphosphate, d-myo-inositol 1,4,5,6-tetrakisphosphate [Ins(1,4,5,6)P₄], that is produced acutely in response to a receptor-independent process. Thus, infection of intestinal epithelial cells with the enteric pathogen Salmonella, but not with other invasive bacteria, induced a multifold increase in Ins(1,4,5,6)P₄ levels. To define a specific function of Ins(1,4,5,6)P₄, a membrane-permeant, hydrolyzable ester was used to deliver it to the intracellular compartment, where it antagonized epidermal growth factor (EGF)-induced inhibition of calcium-mediated chloride (Cl⁻) secretion (CaMCS) in intestinal epithelia. This EGF function is likely mediated through a phosphoinositide 3-kinase (PtdIns3K)-dependent mechanism because the EGF effects are abolished by wortmannin, and three different membrane-permeant esters of the PtdIns3K product phosphatidylinositol 3,4,5-trisphosphate mimicked the EGF effect on CaMCS. We further demonstrate that Ins(1,4,5,6)P₄ antagonized EGF signaling downstream of PtdIns3K because Ins(1,4,5,6)P₄ interfered with the PtdInsP₃ effect on CaMCS without affecting PtdIns3K activity. Thus, elevation of Ins(1,4,5,6)P₄ in Salmonella-infected epithelia may promote Cl⁻ flux by antagonizing EGF inhibition mediated through PtdIns3K and PtdInsP₃.