〔Abstract〕 Objective To investigate whether the ferroptosis inhibitor ferrostatin-1(fer-1) has a protective effect on acute liver injury(ALI) in mice caused by overdose of acetaminophen(APAP). Methods (1) Survival experiment. The 40 male ICR mice were randomly divided into 4 groups, namely APAP model group, fer-1 pre-treatment group, fer-1 post-treatment group, and N-acetyl-L-cysteine(NAC) post-treatment group. They were observed for 7 days after treatment and their deaths were recorded.(2) The 48 male ICR mice were randomly divided into 0 h group, fer-1 group, APAP model group(1, 4, 24 h), and fer-1 pretreatment group(1, 4, 24 h). The pathological changes of mouse liver were observed by HE staining. The aspartate aminotransferase(AST), alanine aminotransferase(ALT) in mouse serum and oxidative stress indicator malondialdehyde(MDA) in liver homogenate were detected by biochemical instrument. RT-qPCR was used to detect the expressions of ferroptosis marker genes long-chain fatty acid-CoA synthase 4(ACSL4), prostaglandin endoperoxidase 2(ptgs2), iron metabolism-related genes transferrin receptor 1(TFR1), hepcidin regulatory protein(HJV), transferrin receptor 2(TFR2) and iron regulatory protein 1(IRP1). Results Compared with NAC post-treatment, fer-1 pretreatment and post-treatment could improve the survival rate of mice; compared with the model group, in the fer-1 pretreatment group, the liver coefficient of mice decreased, liver function indicators ALT, AST improved, lipid peroxidation index MDA decreased, ferroptosis gene expression decreased and iron metabolism disorders were ameliorated. Conclusion Fer-1 pretreatment can alleviate ALI induced by APAP in mice. The mechanism may be through inhibiting ferroptosis and improving the expression of iron metabolism-related genes in the liver of mice.