In contrast to other enzymes, Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) has a broad substrate specificity and high catalytic rate when transferred in human cells. This makes it a promising therapeutic agent when administered together with several cytotoxic nucleoside analogs, such as gemcitabine 2',2'-difluoro-deoxycytidine (dFdC). Therefore, lentiviral vectors, which potentially allow stable long-term transgene expression, are good candidates for gene delivery vehicles. In the present study, we successfully developed a lentivirus-mediated transgene expression system of Dm-dNK under the control of hTERT promoter against the breast cancer cell line (Bcap37), the gastric cancer cell line (SGC7901) and the normal fibroblast cell line (WI-38). Moreover, we also analyzed its targeted cytotoxicity in vitro with treatment of the prodrug dFdC. Bcap37 tumor growth was inhibited in nude mice. Both cancer cell lines exhibited apparent cytotoxicity when infected with recombinant lentivirus constructs expressing Dm-dNK. In contrast, lentivirus-infected WI-38 cells exhibited less cytotoxicity. These data suggested that Dm-dNK was sensitive to dFdC, and it resulted in synergistic growth inhibition and apoptosis induction in vitro. In addition, Lenti-hTERT-dNK/dFdC also suppressed tumor growth in vivo. Our results suggest that the Lenti-hTERT-dNK/dFdC system is a safe and feasible treatment strategy in the development of suicide gene therapy.