Signal transducer and activator of transcription 3 (STAT3) and hexokinase 2 (HK2) are involved in hepatocellular carcinoma (HCC). Deregulation of cellular energetics involving an increase in glycolysis is a characteristic of HCC. This study examined whether STAT3 regulates HCC glycolysis through the HK2 pathway in HCC cells. Human HCC cell lines HepG2 and Hep3B cells were transfected with pcDNA3.1(+)-EGFP-STAT3, STAT3 siRNA and HK2 siRNA, respectively, or treated with rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), and the effects on STAT3 and HK2 expression and cell glycolysis were determined. STAT3 and HK2 expressions were evaluated by real-time polymerase chain reaction and Western blotting. The level of glycolysis metabolism was assessed by the determination of glucose consumption and lactate production. The results showed that transfection of HepG2 and Hep3B cells with pcDNA3.1(+)-EGFP-STAT3 significantly increased STAT3 mRNA and protein expression, glucose consumption and lactate production, and HK2 mRNA and protein expression. However, transfection of HepG2 and Hep3B cells with STAT3 siRNA significantly decreased glucose consumption and lactate production and HK2 mRNA and protein expression. Transfection of HepG2 and Hep3B cells with HK2 siRNA significantly decreased glucose consumption and lactate production. Treatment of HepG2 and Hep3B cells with rapamycin significantly reduced HK2 mRNA and protein expression and glucose consumption and lactate production. These results suggest that mTOR-STAT3-HK2 pathway is involved in the glycolysis of HCC cells and STAT3 may regulate HCC glycolysis through HK2 pathway, providing potential multiple therapeutic targets through intervention of glycolysis for the treatment of HCC.