This study aimed to investigate the effect and mechanism of 8-methoxypsoralen (8-MOP) on acetaminophen (APAP)-induced hepatotoxicity in mice. The study found that 1 h after intraperitoneal injection of 300 mg/kg APAP, treatment with 40 mg/kg, 80 mg/kg and 120 mg/kg 8-MOP could reduce serum transaminase level and histopathological liver necrosis area. Elevated mRNA expression of liver inflammatory mediators caused by excessive APAP was also reversed. 8-MOP significantly reduced APAP-induced hepatotoxicity dose-dependently, and the highest therapeutic dose of 8-MOP (120 mg/kg) had no harmful effects on the liver. Cocktail probe assay revealed that 8-MOP can inhibit Cyp2e1 enzymatic activities of mice, thereby reducing the production of acetaminophen-cysteine (APAP-CYS), a toxic metabolite of APAP. 8-MOP had no significant effect on the protein and gene expression of Cyp2e1. The three-dimensional structures of mouse Cyp2e1 were constructed by homologous modeling. Molecular docking showed that 8-MOP had a good binding effect on the enzyme activity site of Cyp2e1. In summary, 8-MOP dose-dependently attenuated APAP-induced hepatotoxicity by binding to Cyp2e1 and occupying the active center of the enzyme, thus competitively inhibiting the oxidative metabolism of APAP, and reducing the generation of toxic product APAP-CYS.