Previously, we demonstrated that signal transducer and activator of transcription factor 1 (STAT1) plays an essential role in liver injury induced by lipopolysaccharide (LPS)/D-galactosamine (D-GalN); however, the underlying mechanism involved remains unclear. Here, we showed that LPS/D-GalN administration induced secretion of tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma), which mediated apoptosis synergistically. Moreover, LPS/D-GalN-induced apoptosis was associated with increased inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production, as well as elevated reactive oxygen species (ROS) production, which were all strongly inhibited by treatment with the antioxidant N-acetyl-L-cysteine (NAC) and an iNOS/NO inhibitor, L-NMMA. Although STAT1 activation and expression did not change significantly in TNF-alpha/IFN-gamma-cotreated cells compared with cells treated with IFN-gamma alone, the absence of STAT1 or interferon regulatory factor 1 (IRF-1) in genetic knockout mice strongly abrogated the observed effects of TNF-alpha/IFN-gamma on iNOS/NO induction, ROS production, loss of mitochondrial transmembrane potential (DeltaPsim), and apoptosis compared with STAT1(+/+) and IRF-1(+/+) mice. Additionally, the synergistic effects of TNF-alpha/IFN-gamma on iNOS/NO induction, ROS production, and apoptosis were significantly inhibited by overexpression of dominant negative STAT1 in contrast to overexpression of wild-type STAT1. In STAT1-deficient mice, nuclear factor kappaB (NF-kappaB) activation by TNF-alpha/IFN-gamma was attenuated and strongly inhibited by both NAC and L-NMMA. Moreover, the proteasome inhibitor, MG132, inhibited NF-kappaB activation and strongly inhibited iNOS/NO induction, ROS production, and loss of DeltaPsim induced by TNF-alpha/IFN-gamma, thereby inhibiting apoptosis. Interestingly, it appears peroxynitrite, which is produced by TNF-alpha/IFN-gamma, may interfere with STAT1 phosphorylation by inducing STAT1 nitration. Collectively, these findings demonstrate that TNF-alpha/IFN-gamma synergistically potentiates iNOS/NO induction, ROS production, and loss of DeltaPsim via STAT1 overexpression, playing an important role in promoting apoptosis and liver injury induced by LPS/D-GalN.