Objective:To investigate the killing effect of lipofection-mediated herpes simplex virus-thymidine kinase (HSV-TK) gene combined with hyperthermia on hepatocellular carcinoma HepG2 cell line in vitro. Methods :HepG2 cells were transfected with HSV-TK gene by lipofection and used for in vitro study. The killing effect of GCV combined with hyperthermia on HepG2/tk cells was observed. Results:The cell apoptotic rate of the hyperthermia combined with GCV treatment group was significantly higher than the control group and the single GCV treatment group (P<0.05). As the temperature increased, the effect was more and more obvious. The proliferation inhibition rate was (78.8±7.5)% and apoptotic rate of HepG2/tk cells reached (42.8±3.6)% when treated by hyperthermia at 44 ℃. Conclusions:HSV-TK gene was transfected into HepG2 cells by lipofection and expressed stability. Hyperthermia can significantly enhance the sensitivity of killing effect of GCV on HepG2/tk cells.