The aim of this study was to use gene therapy via the Sleeping Beauty (SB) system to increase telomerase promoter activity to target hepatocellular carcinoma (HCC). In previous studies, we identified selective and increased expression of luciferase and suicide genes controlled by the hTERT (human telomerase reverse transcriptase) promoter and the SV40 enhancer in telomerase-positive cancer cell lines. Because telomerase is activated in about 80% of HCCs, it is likely that increasing the activity of the telomerase promoter with a suicide gene will effectively eradicate HCCs. We found that the telomerase promoter mediated SB system can efficiently insert transgene into HCC genomes. Also, telomerase promoter activity was increased using a SB vector expressing suicide gene HSV-TK (herpes simplex virus thymidine kinase) controlled by the hTERT promoter and a SV40 enhancer for the induction of telomerase-positive cancer-specific cell death. HCC cell lines transfected with pT.hTp.HSV-tk.Con with active helper plasmid and ganciclovir (GCV) significantly inhibited cancer cell growth. These results indicate that Sleeping Beauty transposon mediated suicide gene expression can be used in HCC-targeted cancer gene therapy.