Objective To establish a mouse model of acute liver failure induced by lipopolysaccharide/D-galactosamine (LPS/D-GalN). Methods The optimum dose of LPS/D-GalN was determined by i.p. injection of eight different doses of LPS and D-GalN into 40 female C57BL/6 mice and observation of their survival time. Then, 32 female C57BL/6 mice were i.p. injected with the optimal dose of LPS/D-GalN and sacrificed at 0, 1, 4, 8 hours after the injection, 8 mice in each group. The control mice received saline injection. Hepatic changes were observed by pathology and serum ALT, IL-6, MCP-1 and TNF-α were measured by biochemistry or flow cytometry. Results LPS (2.5 mg/kg) and D-GalN (0.3 g/kg) were determined as the optimal dose for the establishment of mouse model of acute liver injury. Compared with the control group, the hepatocellular damages were progressing in a positive correlation with the time course after LPS/D-GalN administration. The level of serum ALT was significantly increased after LPS/D-GalN administration(P<0.001). The levels of inflammatory cytokines IL-6, MCP-1 and TNF-α were increased and reached a peak at one hour after LPS/D-GalN administration and then decreased almost to that of the control group 8 hours later(P<0.001). Conclusions The mouse model of acute liver injury is successfully established by LPS/D-GalN administration, and provide an effective animal model for the study of pathogenic mechanisms of acute liver failure and evaluation of therapeutic drugs.