Sestrins ( Sesns) are a family of stress-sensitive genes that have been suggested to regulate lipid metabolism. Chronic ethanol feeding is known to cause lipid accumulation in hepatocytes. This study was designed to investigate the role of S esn3 in the pathogenesis of alcohol-induced hepatic steatosis. We demonstrated that ethanol inhibited the expression of Sesn3 in VL-17A cells. Overexpression of Sesn3 ameliorated triglyceride accumulation; downregulation using short hairpin RNA significantly deteriorated triglyceride accumulation in these cells. The expression of Sesn3 was also reduced in mice fed with ethanol for 4 wk. Overexpression of Sesn3 prevented hepatic steatosis, whereas knockdown of Sesn3 worsened hepatic steatosis in ethanol-fed mice. Overexpression of Sesn3 significantly reduced the expression of genes encoding for lipid synthesis through AMPK pathway. Overexpression of Sesn3 augmented the effect of ethanol on phospho-p70 S6 kinase. The levels of hepatic light chain 3, a marker for autophagy, expression were significantly decreased in ethanol-fed mice after Sesn3 gene was knocked down. Our findings suggest that inhibitory effect of ethanol on Sesn3 may play an important role in the development of ethanol-induced fatty liver.