Apoptosis plays an important role in maintaining the balance between proliferation and cell loss in the intestinal epithelium. Apoptosis rates may increase in intestinal pathologies such as inflammatory bowel disease and necrotizing enterocolitis, suggesting pharmacological prevention of apoptosis as a therapy for these conditions. Here we explore the feasibility of this approach using the rat epithelial cell line IEC-6 as a model. Based on the known role of K⁺ efflux in apoptosis in various cell types, we hypothesized that K⁺ efflux is essential for apoptosis in enterocytes and that pharmacological blockade of this efflux would inhibit apoptosis. Probing the intracellular [K⁺] with K⁺-sensitive fluorescent dye PBFI and measuring efflux of ⁸⁶Rb⁺, we have found that apoptosis-inducing treatment with the proteasome inhibitor MG-132 leads to two-fold increase in K⁺ efflux from IEC-6 cells. Blockade of K⁺ efflux with TEA, 4-AP, stromatoxin, chromanol 293B and the recently described K⁺ channel inhibitor 48F10 prevents DNA fragmentation, caspase activation, release of cytochrome c from mitochondria, and loss of mitochondrial membrane potential. Thus, K⁺ efflux occurs early in the apoptotic program and is required for the execution of the later events. Apoptotic K⁺ efflux critically depends on activation of p38 MAP kinase. These results demonstrate for the first time the requirement of K⁺ channel mediated K⁺ efflux for progression of apoptosis in enterocytes and suggest the use of K⁺ channel blockers to prevent apoptotic cell loss occurring in the intestinal pathologies.