Abstract: Objective 　To investigate the anti-tumor effects of replication-competent adenovirus expressing short interference RNA targeting Ki67 gene ( Ki67-siRNA) and the expression level of Ki67 gene on lung cancer A549 cells in vitro. Methods 　The recombined adenoviruses (ZD55-Ki67), extracted DNA, were verified by PCR. Viruses were propagated on HEK293 cells and purified by CsCI gradient according to standard techniques, and functional PFU titers were determined by plaque assay on 293 cells. The antitumor potential of ZD55-Ki67 to A549 cells was evaluated by fluorescence microscopy、MTT assay and crystal violet dye method. The expression of E1A was also detected by West-blot . The effect of ZD55-Ki67 on the Ki-67 expression of A549 cells was detected by RT-PCR. Results 　The analysis of PCR indicated the recombinant adenovirus ZD55-Ki67 containing Ki67-siRNA gene and without wild adenovirus.Functional PFU titers of ZD55-Ki67 were 4 ×10¹⁰PFU/ ml. West-blot analyses indicated ZD55-Ki67 can express E1A in adenovirus-infected A549 cells. The expression of Ki67-siRNA gene delivered by ZD55-Ki67 is more effective to inhibit the proliferation and the Ki67 expression in mRNA levels of A549 cells than Ad-Ki67 in vitro. Conclusion 　ZD55-Ki67 can inhibit the expression of Ki67 gene and proliferation of lung cancer A549 cells. ZD55-Ki67 could be a powerful tool in further investigations of Ki67 gene and a novel therapeutic st rategy for lung cancer.