The present study was designed to investigate the protective effect of aloin against alcoholic liver disease in a chronic alcohol feeding mouse model. Mice were given alcohol twice a day by intragastric administration for 11 weeks (4.0, 4.7, 5.5 g/kg bw/day for the first 3 weeks respectively, 6.3 g/kg bw/day for the following 8 weeks). Aloin (10, 30 mg/kg bw) or vehicle was given by gavage to mice after each alcohol administration. Alcohol elevated the serum transaminases alanine aminotransferase, aspartate aminotransferase, total cholesterol and triglyceride levels which were significantly attenuated by the co-administration of aloin (p < 0.05). Histopathological observations were consistent with these indices. Co-administration of aloin significantly suppressed the alcohol-dependent induction of sterol regulatory element-binding protein-1c expression (p < 0.01) and remarkably up-regulated the mRNA levels of AMP-activated protein kinase-α2 (p < 0.001). Furthermore, aloin supplementation significantly inhibited the alcohol-dependent elevation of malondialdehyde and cytochrome P4502E1 expression (p < 0.05), and significantly elevated superoxide dismutase activity (p < 0.01). The up-regulation of serum lipopolysaccharide (LPS), hepatic nitric oxide, tumor necrosis factor α, toll-like receptor-4, and myeloid differentiation primary response gene 88 were also markedly suppressed by the co-administration of aloin (p < 0.05) in alcohol-treated mice. These results suggest that aloin may represent a novel, protective strategy against chronic alcoholic liver injury by attenuating lipid accumulation, oxidative stress and LPS-induced inflammatory response.