Abstract: Objective: To investigate the selective killing effects of adenovirus (Ads) mediated double suicide gene (CD/TK) stimulated by vascular endothelial growth factor(VEGF) promoter on breast cancer cells MCF-7. Methods: MCF-7 cells with VEGF expression and normal human mammary epithelial cells without VEGF expression in primary culture were infected by the Ad-VEGFP-CD/TK. The infection efficiency were observed by a fluorescence microscope. The expression of CD/TK was detected by RT-PCR and Western blot. After treatment with GCV and 5-FC, MTT was employed to evaluate the killing effects. Then pathological features were observed by electron microscope and DNA content in MCF- 7 cells was detected by flow cytometry. The caspase-3 activity was detected by absorption spectrometry. Results: The infection rates of the resultant recombinant Ads to MCF-7 and human mammary epithelial cells were not apparently different, and the rates increased gradually with the addition of multiplicity of infection(MOI) of Ads. RT-PCR and Western blot demonstrated that the product of CD/TK gene existed in MCF-7 cells infected by Ad-VEGFP-CDTK, but not in infected human mammary epithelial cells. MTT assay showed that MCF-7 cells infected with Ads were highly sensitive to the prodrugs, while the infected human mammary epithelial cells were not. At the MOI of 100, morphologic features of apoptosis in MCF-7 cells were observed by electron microscope. The pro-drug administered group showed an apoptotic peak in flow cytometry. Furthermore, the activity of caspase-3 gradually increased with the increasing concentration of the pro-drugs. Conclusion: The CD/TK fusion gene system controlled by VEGF promoter has selective killing effects on MCF-7 cells with VEGF expression and induces apoptosis. The increased activity of caspase -3 may be one of the mechanisms for MCF-7 cell apoptosis induced by pro-drugs.