Abstract: Objective To construct the recombinant adenovirus vector expressing survivin shRNA,which mediates siRNA to inhibit the proliferation of human lung cancer A549 cells. Methods Cotransfection of pShuttle -survivin and adenovirus plasmid with LacZ tag into HEK-293 T cells was conducted to get recombinant adenovirus.Virus titer was determined after verification by PCR. Infection efficiency of the adenovirus was investigated by β-gal staining.MTT and FCM were employed to study its biological effects,RT-PCR was used to estimate the effects of Ad-survivin on reducing the expression of survivin in A549 cells.Hoechst staining was used to verify whether cell apoptosis could be induced or not. Results Ad-survivin was constructed successfully and its infection efficiency on A549 cells was evident.By means of Ad-survivin,the proliferation of A549 cells were suppressed and cells were blocked at the G_2/M phase.RT-PCR results showed that the expression of survivin mRNA was repressed by Ad-survivin,and Hoechst staining indicated that knockdown of survivin expression by RNAi induced apoptosis in A549 cells. Conclusion Recombinant adenovirus with shRNA targeting survivin has been successfully constructed and its tumor suppression in A549 cells was detected,which may provide an effective way of gene therapy on human lung cancer.