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 α (TNF-α) and interferon γ (IFN-γ), 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-α/IFN-γ-cotreated cells compared with cells treated with IFN-γ alone, the absence of STAT1 or interferon regulatory factor 1 (IRF-1) in genetic knockout mice strongly abrogated the observed effects of TNF-α/IFN-γ on iNOS/NO induction, ROS production, loss of mitochondrial transmembrane potential (ΔΨm), and apoptosis compared with STAT1(+/+) and IRF-1(+/+) mice. Additionally, the synergistic effects of TNF-α/IFN-γ 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 κB (NF-κB) activation by TNF-α/IFN-γ was attenuated and strongly inhibited by both NAC and l-NMMA. Moreover, the proteasome inhibitor, MG132, inhibited NF-κB activation and strongly inhibited iNOS/NO induction, ROS production, and loss of ΔΨm induced by TNF-α/IFN-γ, thereby inhibiting apoptosis. Interestingly, it appears peroxynitrite, which is produced by TNF-α/IFN-γ, may interfere with STAT1 phosphorylation by inducing STAT1 nitration. Collectively, these findings demonstrate that TNF-α/IFN-γ synergistically potentiates iNOS/NO induction, ROS production, and loss of ΔΨm via STAT1 overexpression, playing an important role in promoting apoptosis and liver injury induced by LPS/d-GalN.