In our present study, we took advantage of the characteristics of RNA interference technology, which can efficiently, stably, and specifically silence target genes, and designed a small interfering RNA (siRNA) that could specifically target hTERT mRNA. We used a lentiviral vector (LV) to deliver the hTERT siRNA into telomerase-positive A549 lung cancer cells and investigated the effect of hTERT siRNA on the hTERT mRNA levels, hTERT protein levels, cell proliferation, and apoptosis in the lung cancer cells. The results from quantitative PCR, Western blotting, and the MTT assay showed that the expression levels of both hTERT mRNA and protein in the cells were significantly decreased and that the cell proliferation rate started to significantly slow down at 48 h after transfection with hTERT-LV. Our study demonstrated that siRNA sequences specifically targeting hTERT mRNA, which were packaged into lentivirus particles and then used to transfect the lung cancer cell line A549, can specifically silence the mRNA of the target gene, hTERT, and then reduce the hTERT protein expression level, which, in turn, reduces cell proliferation, inhibits cell growth, and induces apoptosis.