HIV-1 derived lentiviral vectors (LVs) have emerged as a powerful tool for gene delivery. hTERT is an ideal tumor-associated antigen with which to develop a potential dendritic cell (DC) vaccine. The purpose of this study was to construct a recombinant lentivirus vector of the hTERT peptide and to determine the hTERT-specific cytotoxic T lymphocyte response elicited by DCs transfected with hTERT-lentivirus vectors in vitro. LVs encoding the hTERT peptide were constructed, DCs from cord blood were prepared, their morphology was observed and phenotype was analyzed by flow cytometry. Lenti-hTERT was transfected into DCs to construct the DC vaccines. T lymphocytes stimulated with DC vaccines and HepG2 cells (hTERT+) or 293T cells (hTERT-) were co-cultured for 24 h, respectively. The ability to stimulate proliferation of allogeneic T lymphocytes and the killing activity of CTLs activated by these DCs were determined using the MTT method. According to our results, the recombinant vector lenti-hTERT and lenti-hTERT-DC vaccine were successfully constructed. The stimulatory capacity of the lenti-hTERT DCs in the allogeneic T lymphocyte reaction was markedly enhanced compared with the DC control group (P<0.01). Inhibition rates in HepG2 cells of CTLs stimulated with lenti-hTERT-DCs (CTLT) were significantly higher than CTLs stimulated with the control DC group (CTLN) (P<0.01). Inhibition rates in 293T cells of CTLT and CTLN were low and there was no difference between the different DC groups (P>0.05). DCs transfected with the hTERT peptide were capable of eliciting a stronger hTERT-specific CTL response in vitro. Our data indicate that lenti-hTERT-DCs may potentially be used as an effective approach for cancer immunotherapy.