Glucosamine (GLC) attenuates alcohol-induced acute liver injury by inhibiting oxidative stress and inflammation. This study investigated the protective effects and mechanisms of GLC on alcohol-induced liver injury in mice and human hepatocytes (L02 cells). Mice were administered GLC for 30 days, while L02 cells were exposed to ethanol to induce liver injury. The levels of liver injury biomarkers, lipid metabolism, oxidative stress markers, and inflammatory factors were measured. Western blot and RT-PCR were used to explore oxidative and inflammatory pathways in mouse liver tissue. Results showed that GLC significantly reduced the levels of AST, ALT, ALP, LDH, TG, TC, VLDL, and LDL-C, while increasing HDL-C. GLC also reduced MDA levels and increased GSH, CAT, and SOD levels, indicating reduced oxidative stress. Mechanistically, GLC inhibited the expression of CYP2E1, activated the Keap1/Nrf2/HO-1 antioxidant pathway, and downregulated the phosphorylation of MAPK and NF-κB pathways, thereby reducing the expression of TNF-α, IL-1β, and IL-6. These findings suggest that GLC may serve as a significant candidate functional food for attenuating alcohol-induced acute liver injury. The study provides a theoretical basis for the development of dietary supplements to prevent alcoholic liver disease (ALD). GLC is a safe and non-toxic dietary supplement with anti-inflammatory and antioxidant properties, making it a promising candidate for the treatment of ALD. Further research is needed to explore the specific mechanisms of GLC in lipid metabolism and the role of the gut-liver axis in ALD.Glucosamine (GLC) attenuates alcohol-induced acute liver injury by inhibiting oxidative stress and inflammation. This study investigated the protective effects and mechanisms of GLC on alcohol-induced liver injury in mice and human hepatocytes (L02 cells). Mice were administered GLC for 30 days, while L02 cells were exposed to ethanol to induce liver injury. The levels of liver injury biomarkers, lipid metabolism, oxidative stress markers, and inflammatory factors were measured. Western blot and RT-PCR were used to explore oxidative and inflammatory pathways in mouse liver tissue. Results showed that GLC significantly reduced the levels of AST, ALT, ALP, LDH, TG, TC, VLDL, and LDL-C, while increasing HDL-C. GLC also reduced MDA levels and increased GSH, CAT, and SOD levels, indicating reduced oxidative stress. Mechanistically, GLC inhibited the expression of CYP2E1, activated the Keap1/Nrf2/HO-1 antioxidant pathway, and downregulated the phosphorylation of MAPK and NF-κB pathways, thereby reducing the expression of TNF-α, IL-1β, and IL-6. These findings suggest that GLC may serve as a significant candidate functional food for attenuating alcohol-induced acute liver injury. The study provides a theoretical basis for the development of dietary supplements to prevent alcoholic liver disease (ALD). GLC is a safe and non-toxic dietary supplement with anti-inflammatory and antioxidant properties, making it a promising candidate for the treatment of ALD. Further research is needed to explore the specific mechanisms of GLC in lipid metabolism and the role of the gut-liver axis in ALD.