Abstract: Objective To evaluate the killing effect of adenovirus (Ad)-mediated double suicide gene driven by kinase domain-containing receptor (KDR) promoter on gastric cancer MGC-803 cells. Methods The 293 packaging cells were transfected by the plasmids pAdEasy-KDR-CDglyTK to generate infectious viruses. The gastric cancer MGC-803 cells were infected by the Ad followed by treatment with 5-FC and/or ganciclovir at different concentrations. The cell-killing effects were evaluated and the bystander effects analyzed after coculture of the cells without AdKDR-CDglyTK infection with the infected cells at different ratios. The cell cycle distribution was detected by flow cytometry and the pathological changes of the cells were observed by electron microscopy. Results The infection rate of the resultant recombinant Ad in the cells increased gradually with increment of the multiplicity of infection (MOI) of the Ads. The killing effect of CD/TK fusion gene on the MGC-803 cells was much stronger than that of either of the single suicide gene (P<0.001), and considerable bystander effect was observed. The Ad infection caused MGC-803 cell growth arrest at G1 phase with onset of apoptotic and necrotic morphologies of the cells as seen under electron microscope. Conclusions The CD/TK fusion gene system driven by the KDR promoter possesses effective killing effect on the KDR-expressing gastric cancer MGC-803 cells.