Background: Genistein is a natural isoflavone with many health benefits, including antitumour effects. Increased hypoxiainducible factor 1 alpha (HIF-1 alpha) levels and glycolysis in tumour cells are associated with an increased risk of mortality, cancer progression, and resistance to therapy. However, the effect of genistein on HIF-1 alpha and glycolysis in hepatocellular carcinoma (HCC) is still unclear. Methods: Cell viability, apoptosis rate, lactate production, and glucose uptake were measured in HCC cell lines with genistein incubation. Lentivirus-expressed glucose transporter 1 (GLUT1) or/and hexokinase 2 (HK2) and siRNA of HIF-1 alpha were used to test the direct target of genistein. Subcutaneous xenograft mouse models were used to measure in vivo efficacy of genistein and its combination with sorafenib. Results: Genistein inhibited aerobic glycolysis and induced mitochondrial apoptosis in HCC cells. Neither inhibitors nor overexpression of HK2 or GLUTs enhance or alleviate this effect. Although stabiliser of HIF-1 alpha reversed the effect of genistein, genistein no longer has effects on HIF-1 alpha siRNA knockdown HCC cells. In addition, genistein enhanced the antitumour effect of sorafenib in sorafenib-resistant HCC cells and HCC-bearing mice. Conclusions: Genistein sensitised aerobic glycolytic HCC cells to apoptosis by directly downregulating HIF-1 alpha, therefore inactivating GLUT1 and HK2 to suppress aerobic glycolysis. The inhibitory effect of genistein on tumour cell growth and glycolysis may help identify effective treatments for HCC patients at advanced stages.