Abstract: Objective To construct adenovirus vector Ad.HRE.CArG.HSV-TK regulated by hypoxia and radiation via AdEasy system and to observe its radiosensitization effect in human breast cancer cell lines Bcap37 and MDA-MB-435.Methods Using molecular clone technologies, Ad.HRE.CArG.HSV-TK inserted with HRE.CArG.HSV-TK fragment was constructed. The virus was purifed by CsCl gradient centrifuge. The functional titer of adenovirus was detected by GFP tag expression. Cell lines of Bcap37 and MDA-MB-435 were transducted with adenovirus in vitro, 48h later, expression rate of GFP was detected by flow cytometry.TK gene mRNA and protein expression were detected with real -time RT-PCR and Western blot respectively.The growth inhibition effect and sensitizer enhancement ratio( SER)of adenovirus（Ad）transduction combined with radiation（RT） were detected with MTT assay.Results The final titers of adenovirus was 2.1×109TU/ml. Bcap37 and MDA-MB-435 cells transducted by 50 MOI adenovirus in vitro for 48h, positive rate of GFP was 92% and 93%,respectively.TK expression increased dramatically（P<0.05）. Growth inhibition rate in group of Ad＋RT was significantly higher than that in Ad group and the same dose of RT group（P<0.05）. SER were both 1.55.Conclusion The recombinant adenovirus expressing GFP and TK gene can be generated via AdEasy system.The adenovirus has radiosensitization effect on breast cancer cells. It may serve as a basis for later gene radiotherapy research in breast cancer.