Abstract: Objective: In this study, the protective effect of ursolic acid (UA) against acute alcohol-induced liver injury in mice and the mechanism involved was explored. Methods: Forty specific-pathogen-free male ICR mice were randomly assigned to control, model, high-dose UA (80 mg/kg), medium-dose UA (40 mg/kg), and low-dose UA groups (20 mg/kg). Changes in liver pathology, expression of proteins in the nuclear factor-kappa B (NF-κB) pathway, and liver metabolomics were analyzed by determining the mouse serum activities of aspartate transaminase (AST) and alanine transaminase (ALT) and levels of total cholesterol (TC), triglyceride (TG), and inflammatory factors interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) as well as the liver tissue activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and level of malondialdehyde (MDA). Results: Compared with the model group, the UA groups showed significantly lower (P<0.01 or P<0.05) serum ALT and AST activities and TC, TG, IL-1β, IL-6, and TNF-α levels. Moreover, in the UA groups, the liver activities of GSH-Px and SOD were significantly increased (P<0.01 or P<0.05), and the levels of the lipid peroxidation marker MDA were significantly decreased (P<0.01). Western blot results showed that the protein expression levels of Toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), NF-κB, and p-NF-κB in the inflammatory pathway were significantly (P<0.01 or P<0.05) reduced in the UA groups. The liver metabolomics results showed significant changes in 19 differential metabolites among the control, model, and UA groups. The levels of the 19 differential metabolites tended to normalize with the action of UA. These differential metabolites were mainly concentrated in pathways of linoleic acid metabolism, ether lipid metabolism, unsaturated fatty acid biosynthesis, glutathione metabolism, and glycerophospholipid metabolism. Conclusion: UA protects mice from acute alcohol-induced liver injury by inhibiting oxidative stress, regulating lipid metabolism, and alleviating inflammatory damage. The mechanism may be related to various metabolic pathways, including those of linoleic acid metabolism, unsaturated fatty acid biosynthesis, glutathione metabolism, and glycerophospholipid metabolism.