Objective: To construct novel RNAi expression cassettes targeting hTERT and hTR, and then observe the effects of hTERT and hTR gene interference alone or in combination on telomerase activity, cell apoptosis and cell cycle in tumor cells, so as to find new strategies for tumor gene therapy. Methods: The RNAi expression cassettes targeting hTERT or hTR gene were synthesized by overlap extension PCR. After identification, the established cassettes were transfected into A549 cells alone or in combination, and then the telomerase activity was detected by TRAP-silver staining and TRAP-qPCR, and cell apoptosis rate and cell cycle were determined by flow cytometry. Results: The novel RNAi expression cassettes targeting hTERT and hTR were successfully constructed. Comparing with blank control group or negative control group, the telomerase activities were significantly reduced, the cell apoptosis rates were significantly increased and the G1 cell cycle arrest was significantly increased in A549 cells after transfection of the cassettes targeting either hTERT or hTR gene, and further, the above changes were more significant in A549 cells after combined transfection of the cassettes targeting hTERT and hTR gene than in those transfected with either of them alone (all P<0.05). Conclusion: The expression cassettes of novel RNAi targeting hTERT and hTR can effectively inhibit the telomerase activity, induce the cell apoptosis and change the cell cycle in A549 cells. Novel RNAi technology based on the artificial miRNA expression cassettes is expected to become a new tool in tumor gene therapy.