Robust evidence suggested that gut-derived lipopolysaccharide (LPS) plays a significant role in various liver injury diseases; however, the role of gut-derived LPS in acetaminophen (APAP) overdose-induced acute liver injury remains unclear. The present study aimed to investigate the effect of gut-derived LPS on APAP-induced liver injury. Our results revealed that reduction of gut-derived LPS using multiple antibiotics could significantly exacerbate APAP-induced liver injury and increase mortality in mice. By contrast, pretreatment with exogenous LPS could reverse APAP-induced liver hepatotoxicity in mice and rats. We observed that TNF-alpha secretion in the liver was significantly increased after LPS pretreatment. In addition, depletion of TNF-alpha or TNFR1 inhibited the protective effects of LPS against APAP-induced hepatotoxicity, which indicated that the TNF-alpha/TNFR1 pathway was required to protect against APAP-induced liver injury. Mechanistically, LPS reduces oxidative stress by upregulating the expression of hepatic GSH, reducing MDA levels in liver tissues, and upregulating the expression of several antioxidant genes after APAP injection. However, the production of hepatic GSH was not enhanced in the liver tissues of rats lacking TNF-alpha, or TNFR1 and MDA levels were not reduced after LPS and APAP co-treatment. The above results suggested LPS alleviated APAP-induced oxidative stress via the TNF-alpha/INFR1 pathway. (C) 2019 Published by Elsevier Inc.