Hepatocellular carcinoma (HCC) is a common malignant carcinoma worldwide and the third leading cause of cancer mortality. However, current treatment strategies are not potent enough to combat this disease. Therefore, identification of novel and more effective treatments is crucial. Of the current methods, gene therapy, which targets cancer‑specific expression and limits toxicity, is a new strategy for treating cancers. In this study, we developed a retroviral vector containing herpes simplex virus type-1-thymidine kinase (HSV1‑TK) and a short hairpin RNA for the human telomerase reverse transcriptase (hTERT) gene and investigated the antitumor effects in an in vitro and in vivo mouse model of liver cancer, monitored by PET image. In vitro experiments on HCC cells in the TK-shTERT treatment group showed significant accumulation of 18F-FHBG, which preferentially inhibits HCC cell growth with extremely limited toxicity in normal cells. In vivo studies showed a significant reduction of growth in the TK-shTERT treatment group. In conclusion, these findings showed that combination HSV1-TK/hTERT gene therapy effectively and safely inhibits HCC cell growth in vitro and in vivo and is worthy of development in clinical trials for the treatment of HCC.