Background and aims <p>Sex-determining region on the Y chromosome (SRY), a male-specific gene, can promote the development of various liver diseases including liver fibrosis, hepatocellular carcinoma. Its role in acute liver injury (ALI), however, remains elusive. Here, we aimed to investigate the effects of SRY on ALI.</p> Methods <p>We constructed hepatocyte-specific <i>Sry</i>-overexpressing transgenic (<i>Sry</i>-TG) and hepatocyte-specific <i>Sry</i> knockout (<i>Sry</i>^ΔHC) mice and subjected them to acetaminophen (APAP) insult to determine the potential function of SRY in ALI. Multiomics analysis was used to explore the molecular mechanisms through which SRY affects ALI.</p> Results <p>SRY expression was markedly upregulated in the livers of both male patients and mice with ALI. <i>Sry</i>-TG mice exhibited significantly exacerbated liver injury and increased apoptosis and oxidative stress. In contrast, the male <i>Sry</i>^ΔHC mice exhibited a protective phenotype. Mechanistically, RNA-seq revealed that Gstm2/3 expression was downregulated in the hepatocytes of <i>Sry</i>-TG mice. Liquid chromatography-tandem mass spectrometry revealed PARP-1 as a candidate protein that interacts with SRY. SRY enhances the nuclear accumulation of PARP-1. PARP-1, in turn, suppresses Gstm2/3 mRNA expression by PARylating C/EBPβ and inhibiting the C/EBPβ-mediated transcriptional activation of Gstm2/3. Finally, virtual screening of compounds revealed that dutasteride can bind to SRY. Experiments revealed that dutasteride inhibits SRY expression and attenuates acute liver injury.</p> Conclusions <p>Our results suggest that greater SRY expression in males may account for the susceptibility of males ALI. Dutasteride binds specifically to SRY and inhibits its expression, making it a promising therapy for the clinical treatment of ALI.</p>