In the present study, a plasmid-mediated siRNA interference vector targeting the hTERT gene was constructed and stably transfected into H1299 lung cancer cells. Using real-time quantitative fluorescent PCR technology, western blotting and flow cytometry-based cell cycle profiling, the silencing effect of this vector and its inhibitory effect on proliferation in lung cancer cells were explored. Based upon the results of our previous study, a pair of siRNA sequences was selected, and a DNA template primer was designed and synthesized. After cloning of the template primer into the promoter of the pGenesil-1.1 expression vector, the constructed interference vector was validated using enzyme digestion and gene sequencing. The recombinant interference vector and empty vector were separately transfected into H1299 lung cancer cells with cationic liposomes, and stable monoclonally transfected cells were obtained after selection with G418. After stable transfection, hTERT mRNA and protein expression levels were detected using real-time RT-PCR technology and western blotting. Using the MTT method and a colony formation assay, the growth and proliferation of the stably transfected lung cancer cells were determined. Changes in the cell cycle profile of the stably transfected lung cancer cells were detected using flow cytometry. An interference vector targeting the hTERT gene (pGenesil.1-hTERT) was successfully constructed. Enzyme digestion and gene sequencing confirmed that the sequence insertion met the criteria of the design. After transfection of H1299 cells with pGenesil.1-hTERT or an empty vector, the stably transfected monoclonal cell lines H1299-pGenesil.1-hTERT and H1299-pGenesil.1 were obtained. Compared to the control cells transfected with the empty vector, the H1299-pGenesil.1-hTERT cells had significantly lower mRNA expression of hTERT (93.97±0.83% inhibition, with P<0.001). The protein expression of hTERT in H1299-pGenesil.1-hTERT cells was significantly lower compared to that in H1299-pGenesil.1 cells. The rate of proliferation of H1299-pGenesil.1-hTERT cells was lower compared to that of H1299-pGenesil.1 lung cancer cells. In H1299-pGenesil.1-hTERT cells, the number of cells in the G1 phase increased by 18.3% (P<0.05) compared to the control group; the number of cells in the S and G2 phases decreased by 10.4 and 7.9%, respectively (P<0.05). A recombinant plasmid that interfered with the expression of the hTERT target gene was successfully constructed. Upon transfection of the recombinant interference plasmid into H1299 lung cancer cells, hTERT mRNA and protein expression were down-regulated effectively, telomerase activity and cell proliferation were inhibited, and the cell cycle profile was altered.