Type I interferons, like IFN, are major immune response regulators produced and released by activated macrophages, dendritic cells and virus-infected cells. Due to their immuno-modulatory functions and their ability to induce cell death in tumors and virus-infected cells they are used therapeutically against cancers, viral infections and auto-immune diseases. However, little is known about the adverse effects of type I interferons on non-diseased tissue. This study examined the effects of IFN on cell death pathways in renal proximal tubular cells. IFN induced apoptosis in LLC-PK1 cells, characterized by activation of caspases-3, -8, and -9, DNA fragmentation and nuclear condensation. IFN also caused mitochondrial depolarization. Effector caspase activation was dependent on caspase-8 and caspase-9 and was attenuated by Cyclosporine A, a potent mitochondrial megapore blocker. In addition to apoptosis, IFN exposure also decreased renal epithelial barrier function which preceded apoptotic cell death. Caspase inhibition did not influence permeability regulation while significantly attenuating and delaying cell death. These results indicate that IFN causes programmed cell death in non-diseased renal epithelial cells. IFN-induced apoptosis is directed by an extrinsic death receptor signaling pathway, amplified by an intrinsic mitochondrial pathway. Caspase-dependent and -independent apoptotic mechanisms are involved. These findings reveal a novel aspect of IFN actions with implications for normal renal function in immune reactions and during IFN therapy.