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We investigated the role of zinc‐finger protein 281 (ZNF281), a novel molecule, in ethanol‐induced hepatocyte senescence and uncovered the potential mechanism. Real‐time PCR, Western blot, immunofluorescence staining, and enzyme‐linked immunosorbent assay were performed to explore the role of ZNF281 in hepatocyte senescence. ZNF281 expression was upregulated in both alcohol‐fed mice livers and ethanol‐treated hepatocytes. Silence of ZNF281 in hepatocytes using siRNA mitigated ethanol‐caused decrease in cell viability and increased release of aspartate aminotransferase, alanine transaminase, and lactate dehydrogenase. ZNF281 siRNA reduced senescence‐associated β‐galactosidase‐positive cells under ethanol exposure, abolished cell cycle arrest at G0/G1 phase, and diminished senescence‐associated secretory phenotype and proinflammatory cytokines (IL‐1β and IL‐6) release. At molecular level, ZNF281 deficiency altered the expression profile of senescence‐associated proteins including p53, p21, p16, high mobility group AT‐hook 1, and phospho‐histone H2A.X and telomerase‐associated regulatory factors including telomerase reverse transcriptase, telomeric repeat binding factor 1 (TRF1), and TRF2. ZNF281 knockdown promoted hepatocyte recovery from ethanol‐induced mitochondrial dysfunction and ROS production, which was correlated with rescuing HK2‐PINK1/Parkin signalling‐mediated mitophagy. Mechanistically, ZNF281 directly bound to 5′‐GGCGGCGGGCGG‐3′ motif within HK2 promoter region and transcriptionally repressed HK2 expression. Systematic ZNF281 knockdown by adeno‐associated virus encoding ZNF281 shRNA protected mice from alcohol feeding‐caused hepatocyte injury and senescence. This study provides a novel factor ZNF281 as a driver of hepatocyte senescence during alcoholic liver disease.

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