27-Hydroxycholesterol (27OHC), an endogenous oxysterol, has been implicated in various physiological processes, including regulation of estrogen receptor activity and lipid metabolism. However, studies on how 27OHC affects the metabolic changes associated with lipogenesis inhibition in the liver remain limited. This study aimed to investigate the systemic effects of 27OHC on hepatocytes through a comparative proteomic analysis of the proteomes in the 27OHC-treated Mus musculus hepatocyte (AML12) cells. Ingenuity Pathway Analysis revealed significant down-regulation of certain metabolic pathways, such as cholesterol biosynthesis and glycolysis, which are highly associated with lipid metabolism, following 27OHC treatment. Furthermore, in vitro biochemical analysis revealed significant inhibition of the expression of genes associated with the mevalonate (MVA) pathway and a decrease in the total cellular cholesterol levels in AML12 cells and primary hepatocytes following 27OHC treatment. In addition, it was observed that 27OHC significantly reduced the transcript levels of critical glycolytic enzymes such as aldolase, phosphofructokinase, and pyruvate kinase. This inhibition resulted in decreased lactate production and extracellular acidification rate, indicating suppression of glycolytic flux. Concurrently, we proved that down-regulation of reactive oxygen species generation and hypoxia-inducible factor 1-alpha (HIF-1α) expression following 27OHC treatment partially contributed to glycolysis inhibition. Overall, we demonstrated the inhibitory effects of 27OHC on the hepatic MVA pathway and glycolysis, revealing a novel mechanism by which 27OHC regulates lipid metabolism. As the accumulation of cholesterol and lipids promotes hepatic fatty liver disease and increased glycolysis contributes to triacylglycerol maturation, the suppressive effects of 27OHC on hepatic lipid and glucose metabolism may contribute to protection against fatty liver development.