The glycolytic phenotype is a dominant metabolic phenomenon in cancer and is reflected in becoming aggressive. Certain hepatocellular carcinoma lack increased glycolysis and prefer to uptake acetate than glucose for metabolism. Autophagy plays a role in preserving energies and nutrients when there is limited external nutrient supply and maintains glucose level of blood though supporting gluconeogenesis in the liver. As the role of autophagy and gluconeogenesis in HCC following the glycolic activity was not clear, we cultured HCC cells with different glycolytic levels in Hank's balanced salt solution (HBSS) to induce autophagy and conducted the activity of gluconeogenesis. Both autophagy and gluconeogenesis were induced in low glycolytic HCC cells (HepG2). In glycolytic Hep3B cells, only autophagy without gluconeogenesis was induced upon starvation. When autophagy was blocked, the level of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) was reduced in HepG2 cells and not in Hep3B. Altogether, we investigated contribution of hepatic gluconeogenesis to the metabolic phenotype of HCC cells and the role of autophagy as a potential mechanism regulating gluconeogenesis in low glycolytic HCC. (C) 2015 Elsevier Inc. All rights reserved.