Abstract: To investigate the hepatoprotective effect of hemp peptide, an acute alcoholic liver injury model was established in C57BL/6 mice, and the mechanisms were examined with respect to lipid metabolism and oxidative stress. H&E and Oil Red O staining were performed to assess hepatic histopathology. Serum and liver biochemical parameters, antioxidant enzymes, and alcohol-metabolizing enzymes were measured using biochemical assay kits. Key lipid metabolism and oxidative stress proteins were analyzed by Western blot. The results showed that compared with the alcoholic liver injury model control group, the high-dose hemp peptide group (1300 mg/kg·bw) exhibited significantly decreased liver indices in mice (P<0.05), with the hemp peptide group showing the maximum reduction of 12.48%. In the high-dose hemp peptide group, serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (TC), and triglycerides (TG) were significantly reduced respectively (P<0.05). The levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8) in serum were significantly reduced respectively (P<0.05). The activities of superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GSH-Px) in liver tissue were significantly increased respectively, while the malondialdehyde (MDA) content was significantly decreased (P<0.05). Mechanistically, CYP2E1 protein expression was significantly inhibited by hemp peptide, resulting in enhanced alcohol metabolism. Meanwhile, downregulation of SREBP-1c expression was observed, which effectively alleviated lipid metabolism disorders. Furthermore, PPARα protein expression was markedly upregulated, leading to accelerated fatty acid oxidation. Histopathological analysis revealed that hemp peptide ameliorated liver injury in mice to varying degrees. Hemp peptide showed significant protective effects against alcohol-induced acute liver injury in mice, which would provide a theoretical foundation for developing hemp peptide functional foods.