--- - |2- The ketotic cows displayed hepatic lipid metabolic disorder and high blood concentration of glucagon. The present study indicates that glucagon activates the AMPK signalling pathway to increase lipid oxidation and VLDL assembly and decrease lipid synthesis in cow hepatocytes, thereby reducing liver fat accumulation. Abstract The ketotic cows displayed hepatic lipid metabolic disorder and high blood concentration of glucagon. Importantly, adenosine monophosphate‐activated protein kinase (AMPK) signaling pathway plays an important role in the hepatic lipid homeostasis. Therefore, the aim of this study was to investigate the effect of glucagon on AMPK pathway and its underlying mechanism on lipid metabolism in cow hepatocytes. Cow hepatocytes were cultured and treated with glucagon and AMPK inhibitor (BML‐275). The results showed that glucagon significantly promoted the expression of glucagon receptor and increased the phosphorylation level and activity of AMPKα. Activated AMPKα increased the expression level and transcriptional activity of peroxisome proliferator‐activated receptor α, which further increased the expression of fatty acid oxidation genes and lipid oxidation. Furthermore, activated AMPKα inhibited the expression level and transcriptional activity of sterol regulatory element binding protein‐1c and carbohydrate response element binding protein, which decreased the expression of lipogenic genes, thereby decreasing lipid synthesis. In addition, glucagon also increased the expression of very‐low‐density lipoprotein (VLDL) assembly to export intracellular triglycerides (TG). Consequently, the content of intracellular TG was significantly decreased in cow hepatocytes. These results indicate that glucagon activates the AMPK signaling pathway to increase lipid oxidation and VLDL assembly and decrease lipid synthesis in cow hepatocytes, thereby reducing liver fat accumulation. - 'Journal of Cellular Physiology, EarlyView. '