Fasting induces mammalian metabolic switch from glucose to fatty acid-derived ketones, resulting in a marked change in blood glucose, triglyceride, free fatty acid and bhydroxybutyrate levels and a phenotype of transient hepatic steatosis. However, the underlying mechanism remains incompletely understood. This study aimed to investigate the role of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), a rate-limiting enzyme in ketogenesis, in fasting-induced ketogenesis and hepatic lipid accumulation. Hepatocyte-specific HMGCS2 knockout (LKO) mice were generated and used to compare the difference in liver metabolic response to fasting between wild-type (WT) and LKO mice. Our findings demonstrate that HMGCS2 is essential in enhanced hepatic ketogenesis during fasting. Dysfunction of HMGCS2 leads to excessive hepatic triglyceride accumulation and severe liver injury possibly via increased liver fatty acid uptake by CD36. Therefore, liver HMGCS2 may represent a key enzyme in the maintenance of liver lipid homeostasis during fasting.