SCD1 is a key enzyme controlling lipid metabolism and a link between its activity and NAFLD has been proposed. Lipophagy is a novel regulatory approach to lipid metabolism regulation, which is involved in the development of NAFLD. However, the possible functional connection between SCD1 and lipophagy in NAFLD remains unknown. To investigate the molecular mechanisms through which SCD1 regulates lipophagy in hepatic steatosis, the model of hepatic steatosis was established by inducing mouse primary hepatocytes with sodium palmitate and feeding C57BL/6 mice with HFD. Our results indicated that sodium palmitate-treated hepatocytes exhibited increased SCD1 expression, AMPK inactivation and defective lipophagy. Inhibition of SCD1 expression in hepatocytes resulted in enhanced AMPK activity and lipophagy, and reduced lipid deposition. Although SCD1 overexpression led to decreased AMPK activity and lipophagy, lipid deposition was increased in hepatocytes. SCD1 regulated lipophagy through AMPK to affect lipid metabolism in mouse primary hepatocytes. Additionally, compared to HFD-fed mice, CAY10566(an SCD1-specific inhibitor)-treated mice exhibited significantly decreased hepatic steatosis and hepatic lipid droplet accumulation, as well as enhanced AMPK activity and lipophagy. This study elucidated that SCD1 inhibition ameliorates hepatic steatosis by inducing AMPK-mediated lipophagy, suggesting that the SCD1-AMPK-lipophagy pathway is a potential therapeutic target for NAFLD.