Perforin-deficient NOD mice are protected from diabetes, suggesting that cytotoxic granule contents of CD8⁺ T-cells have a significant role in killing β-cells. Despite this, cytotoxic granule effects on human or mouse pancreatic islets have not been reported. We tested the susceptibility of human and mouse islet cells to purified recombinant perforin and granzyme B and measured apoptotic death using a number of assays. Perforin and granzyme B impaired insulin secretion from islet cells, and this was accompanied by cytochrome c release, caspase activation, and DNA fragmentation. Granzyme B–mediated apoptotic changes only occurred in the presence of perforin. When compared with hemopoietic cells, traditionally used as targets to measure cytotoxic T-cell function in vitro, islet cells were relatively resistant to perforin and granzyme B. Inhibition of caspases prevented DNA fragmentation but not cytochrome c release, indicating that mitochondrial disruption due to granzyme B is independent of caspase activation. Consistent with this, islet cells from mice deficient in the BH3-only protein Bid were resistant to cytochrome c release and were protected from apoptosis after exposure to perforin/granzyme B. Our data suggest that Bid cleavage by granzyme B precedes mitochondrial disruption and apoptosis in pancreatic islets.