Abstract: OBJECTIVE To study the effect of ergosterone on acute liver injury in mice induced by alcohol and its effect on the composition of gut microbiota. METHODS C57BL/6 mice were randomly divided into blank group, model group, diammonium glycyrrhizinate group(60 mg·kg^-1), low dose of ergosterone group(30 mg·kg^-1) and high dose of ergosterone group (60 mg·kg^-1), 10 in each group. Except for the blank group and the model group, the other groups were administrated with the corresponding dose for 10 d. On day 9-10, the other groups except the blank group were administered with two doses of 50% alcohol(10 mL·kg^-1) at 12 h intervals to induce a model. Levels of aspartate aminotransferase(AST), γ-glutamyl transpeptidase (γ-GT) in serum and alanine aminotransferase(ALT), prealbumin(PA), malondialdehyde(MDA), superoxide dismutase(SOD) in liver were detected by ELISA. Hematoxylin-eosin staining was used to observe liver tissue pathological changes, and the effect of ergosterone on mice intestinal flora was analyzed by 16S rRNA gene sequencing analysis technology. RESULTS Ergosterone could significantly reduce the levels of AST, γ-GT in serum and ALT, MDA in liver tissue, and significantly increase the levels of PA and SOD in liver tissue. Ergosterone also could regulate the alcohol-induced changes gut microbiota in phylum and genus levels. CONCLUSION Ergosterone has a protective effect on alcohol-induced acute liver injury in mice, and can improve liver injury by regulating the gut microbiota.