Dysregulation of programmed cell death due to abnormal expression of Bcl-2 proteins is implicated in cancer, neurodegenerative diseases, and heart failure. Among Bcl-2 family members, BNip proteins uniquely stimulate cell death with features of both apoptosis and necrosis. Localization of these factors to mitochondria and endoplasmic reticulum (ER) provides additional complexity. Previously, we observed regulation of intracellular calcium stores by reticular Nix. Here, we report effects of Nix targeting to mitochondria or ER on cell death pathways and heart failure progression. Nix-deficient fibroblasts expressing mitochondrial-directed or ER-directed Nix mutants exhibited similar cytochrome c release, caspase activation, annexin V and TUNEL labeling, and cell death. ER-Nix cells, but not mitochondrial-Nix cells, showed dissipation of mitochondrial inner membrane potential, Deltapsi(m), and were protected from cell death by cyclosporine A or ppif ablation, implicating the mitochondrial permeability transition pore (MPTP). ER-Nix cells were not protected from death by caspase inhibition or combined ablation of Bax and Bak. Combined inhibition of caspases and the MPTP fully protected against Nix-mediated cell death. To determine the role of the dual pathways in heart failure, mice conditionally overexpressing Nix or Nix mutants in hearts were created. Cardiomyocte death caused by mitochondrial- and ER-directed Nix was equivalent, but ppif ablation fully protected only ER-Nix. Thus, Nix stimulates dual autonomous death pathways, determined by its subcellular localization. Mitochondrial Nix activates Bax/Bak- and caspase-dependent apoptosis, whereas ER-Nix activates Bax/Bak-independent, MPTP-dependent necrosis. Complete protection against programmed cell death mediated by Nix and related factors can be achieved by simultaneous inhibition of both pathways.