Background: Acetaminophen (APAP) hepatotoxicity is associated with a high rate of gram- negative enteric bacterial infection; however, the underlying mechanism is still unknown. APAP overdose induces massive hepatocyte necrosis, necrotic tissue releases high mobility group B1 (HMGB1) and exogenous HMGB1 is able to induce gut bacterial translocation (BT) in normal mice; therefore, it is possible that HMGB1 mediates gut BT in APAP hepatotoxicity. This study aims to test this hypothesis by using anti-HMGB1 neutralizing antibody to treat APAP overdose for 24-48 hours. Methods: Male C57BL/6 mice were intraperitoneally (i.p.) injected with a single dose of APAP (350 mg/ kg dissolved in 1 mL sterile saline). 2 hrs after APAP injection, the APAP challenged mice were randomized to receive treatment with either anti-HMGB1 antibody (400 mu g per dose) or non-immune (sham) IgG every 24 h for a total of 2 doses. Results: 24 and 48 hrs after APAP challenge, anti-HMGB1 treatment instead of sham IgG therapy significantly decreased serum HMGB1 concentrations and reduced BT by 85%; serum HMGB1 levels were positively correlated with the amount of BT; anti-HMGB1 therapy decreased hepatic BT at 48 h, which was associated with better recovered liver structure and better restored hepatic immune system that was shown by enhanced hepatic mRNA expression of TNF-alpha, IL-6 and extensive proliferation of inflammatory and reticuloendothelial cells; however, anti- HMGB1 treatment did not decrease gut mucosal permeability as compared to the sham IgG therapy at either 24 or 48 hrs. Conclusion: HMGB1 neutralization is associated with bacterial translocation during APAP hepatotoxicity.