The aim of this study was to investigate the possible protective effects of melatonin and β-D-glucan against AA-induced liver injury in rats. Forty (Spraque–Dawley male) rats were randomly divided into 5 experimental groups: sham (S), acetaminophen only (AA, 900 mg/kg), melatonin (10 mg/kg) + AA (MLT), βD-glucan (50 mg/kg) + AA (β), and melatonin + β-D-glucan + AA (MLT+β) groups. All of the rats were killed on day 11 of the experiment. Histopathological changes and biochemical parameters including levels of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and liver tissue malondialdehyde (MDA), activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were determined to assess the liver function. MDA levels were the highest in the AA group whereas activities of SOD, CAT, and GPx in the liver tissue were found as lowest in this group. MDA activities were significantly lower in the MLT+β group than in the AA group. Only GPx activities in the MLT+β group were significantly higher than those in the MLT and β groups. The serum AST and ALT levels were increased significantly following treatment with AA (p < 0.001). Pretreatment with the antioxidant compounds decreased AST levels significantly but again, the levels were still significantly higher than the sham levels (p < 0.001). There were no statistically significant differences in the microscopic damage between the S, MLT, β, and MLT+β groups (p > 0.05). We concluded that combination of melatonin and β-D-glucan may be attributed to scavenging free radicals and stimulating the antioxidant enzymes