Abstract: Background and purpose: Hepatocellular carcinoma is the third most common malignant tumor worldwide. Cancer stem cells are considered to be the seeds of tumor recurrence, while Warburg effect refers to the aerobic glycolysis characteristic of tumor cells. This study aimed to investigate the relationship between tumor cell stemness and cell glucose metabolism characteristics. Methods: Flow cytometry was used to sort the tumor cells with high expression of stem cell markers in liver tumor cell lines Huh-7 and MHCC97H for serum-free suspension culture, and its morphological changes were observed. The expression levels of stem cell markers and glycolysis enzymes, the glucose uptake capacity and lactic acid production ability of the two cell lines were compared. Results: Cancer stem cells became spheroid in a serum-free suspension culture system. Compared with non-stem tumor cells, the expression levels of key glycolysis enzymes and stem cell markers in spheroid cells were significantly increased (P<0.05). The ability to produce lactic acid was also significantly enhanced (P<0.05), and the stemness of tumor cells was related to the activation of glycolysis. Conclusion: Serum-free suspension culture can be used to enrich cancer stem cells. Compared with non-stem tumor cells, the transformed degree of glucose metabolism in tumor stem cells is stronger. There is a relationship between the stemness of tumor cells and the activation of glycolytic pathway.