〔Abstract〕 Objective To used Toll-like receptor 4(TLR4)inhibitor(TAK-242) investigate the role of TLR4 in the early stage of acetaminophen(APAP)-induced acute liver injury in mice. Methods Fifty-eight male institute of cancer research(ICR) mice were randomly divided into control group(normal control group, solvent control group, inhibitor control group) and experimental group(APAP 4-hour group, APAP+TAK-242 4-hour group, APAP 12-hour group, APAP+TAK-242 12-hour group). Mice in the experimental group were given a single dose of APAP(300 mg/kg) and TAK-242(3 mg/kg) were given intraperitoneal injection 3-hour before APAP injection. The serum alanine aminotransferase(ALT) level and liver index of mice in each group were compared; HE staining showed pathological changes of liver tissue; the level of high mobility group box-1(HMGB1) was determined by immunohistochemistry; the levels of TLR4, HMGB1 and nuclear factor kappa B(NF-κB)protein were detected by Western blot; the levels of monocyte chemoattractant protein-1(MCP-1), interleukin(IL)-1β, tumor necrosis factor-α(TNF-α)and IL-6 genes were determined by real-time fluorescence quantitative PCR(RT-qPCR). The content of IL-6 in liver tissue was determined by enzyme linked immunosorbent assay(ELISA). Results HE staining showed liver tissue of mice obvious swelling and congestion of in APAP group at 4-hour and typical lobular central necrosis in APAP group at 12-hour; the degree of liver necrosis in APAP+TAK-242 groups at 4-hour and 12-hour was less than that in APAP group at the same time point. Compared with the normal control group, serum ALT level, liver index, TLR4, HMGB1, NF-κB protein content, MCP-1, IL-1β, TNF-α, IL-6 gene levels and IL-6 content in liver tissue of APAP 4-hour group increased;serum ALT level, liver index, HMGB1protein content and IL-6 content in liver tissue increased in APAP 12-hour group. Compared with APAP 4-hour group, serum ALT level, liver index, TLR4, HMGB1, NF-κB protein content, MCP-1, IL-1β, TNF-α, IL-6 gene level and IL-6 content in liver tissue decreased in APAP + TAK-242 4-hour group. Compared with APAP 12-hour group, the levels of TLR4, HMGB1 protein and MCP-1, IL-1β, TNF-α genes in APAP + TAK-242 12-hour group decreased.Conclusion Inhibition of TLR4 may inhibit TLR4/HMGB1 pathway to reduce the inflammatory response in the early stage of acetaminophen-induced acute liver injury in mice.