Granzyme F (GzmF) belongs to a unique group of granzymes in mice. Murine GzmF is highly expressed in NK3.1 cells and in lymphokine-activated killer (LAK) cells. However, the manner in which GzmF works in granule-mediated cytolysis is unknown. In this study, we first demonstrated that GzmF causes a novel cell death pathway. The death is characterized by an externalization of phosphatidylserine, by nuclear condensation, mitochondrial damage, cytochrome c (cyt c) release, caspase inactivation and single-stranded DNA nicking. GzmF-induced chromatin was incompletely condensed and segmented at the nuclear periphery. Cellular organelles were damaged and the cytoplasm showed an extensive vacuolization that is reminiscent of necroptosis. GzmF can cause rapid mitochondrial swelling, depolarization and reactive oxygen species accumulation. GzmF-induced death does not involve caspase activation, Bid cleavage or activation of DNA nickase NM23H1. GzmF-silenced LAK cells showed reduced cytotoxicity against caspase-inhibited target tumor cells. Moreover, cyt c release is independent of Bid or Bax/Bak. We further showed that GzmF impairs mitochondrial electron transport to abolish ATP generation. ATP decline may contribute to a failure of apoptosome formation, leading to caspase inactivation.