High-mobility group box 1 (HMGB1) protein is released into the serum after tissue damage, and serves as a warning signal to enhance the inflammatory response. Acute liver injury is one of the diseases that starts with tissue damage and ends with systemic inflammation. We used d-(+)-galactosamine hydrochloride (d-GalN)/lipopolysaccharide (LPS)-treated mice as an acute liver injury model to explore the functions of HMGB1 in more detail. HMGB1 is released into the serum at a very early stage of d-GalN/LPS-induced acute liver injury. It upregulates the expression of tumor necrosis factor-α (TNF-α), interleukin-6, inducible nitric oxide synthase, and tissue factor. TNF-α and HMGB1 form a positive feedback loop to amplify the downstream signals. mAbs against HMGB1 and TNF-α have synergistic effects in protecting mice from d-GalN/LPS-induced acute liver failure. The results suggest that HMGB1 is a key mediator in d-GalN/LPS-induced acute liver injury. Tissue damage and cell necrosis shortly after administration of d-GalN and LPS lead to early HMGB1 release, and HMGB1 acts synergistically with TNF-α to promote pathological processes in acute liver failure.