Mitochondrial cytochrome c release plays a critical role in apoptotic signal cascade after the activation of cell surface death receptors. We investigated the role played by nitric oxide (NO) in mitochondrial apoptotic signaling in tumor necrosis factor α (TNF-α) plus actinomycin D (TNF-α/ActD)-induced apoptosis. NO produced either by S-nitroso-N-acetyl-DL-penicillamine (SNAP) or inducible NO synthase (iNOS) prevented TNF-α/ActD-induced apoptosis in hepatocytes and also inhibited both caspase-8–like (IETDase) and caspase-3–like protease (DEVDase) activity as well as mitochondrial cytochrome c release. Recombinant human (rh) caspase-8 induced the cleavage of the cytochrome c–effluxing factor Bid and cytochrome c release from purified mitochondria in the reconstitution system with Bid +/+ cytosol, but not with Bid −/− cytosol. The addition of SNAP and the caspase-8 inhibitor Ac-IETD-fmk inhibited caspase-8–dependent Bid cleavage and cytochrome c release. The inhibitory effect of NO on caspase-8 was reversed by dithiothreitol (DTT). Furthermore, rh-caspase-8 was found to be modified by S-nitrosylation with 1.7 moles of NO bound per mole of enzyme. Treatment of hepatocytes with interleukin 1β (IL-1β) plus interferon gamma (IFN-γ), which induced iNOS expression and NO production, suppressed TNF-α/ActD-induced Bid cleavage and mitochondrial cytochrome c release. The NOS inhibitor N G-monomethyl-L-arginine (NMA) inhibited the protective effects of IL-1β and IFN-γ. The liver-specific NO donor V-PYRRO/NO also inhibited in vivo elevation of IETDase activity, Bid cleavage, and mitochondrial cytochrome c release in the livers of rats injected with TNF-α plus D-galactosamine. Our results indicate that one mechanism by which NO protects hepatocytes from TNF-α/ActD-induced apoptosis is via the interruption of mitochondrial apoptotic signaling through S-nitrosylation of caspase-8.