Objective: The aim of the study was to construct recombinant type 5 adenovirus expressing the human DBC2(deleted in breast cancer 2) gene for in vitro and in vivo assay in human bladder cancer research. Methods: The human DBC2gene was first subcloned into a shuttle plasmid pAdTrack-CMV. After recombining with pAdEasy-1 vector in BJ5183 cells, thenew recombinant vector pAdEasy-DBC2-CMV was transfected into HEK-293 cells to produce adenovirus. The human bladdercancer cell line T24 was infected with DBC2-containing adenovirus particles. Both RNA and protein were collected from cellsharvested at 72 h after infection. Real time quantitative PCR (qPCR) and Western blot were used to examine mRNA and proteinlevels. Fluorescence microscopy was utilized to observe the expression of reporter green fluorescence protein. Results:Electrophoresis showed there was a 2.2 kb size band produced from high fidelity PCR. Pac I digest of the final producedrecombinant vector yielded band sizes of approximately 30 kb and 4.5 kb. After virus infection with the pAdEasy-DBC2-CMVvector, the T24 cell line was observed to highly express green fluorescence protein under a fluorescence microscope. qPCRand Western blot assay identified that the DBC2 gene was overexpressed at both the mRNA and protein levels in virustransfected cells. Conclusion: By using the pAdEasy adenovirus system, we successfully constructed an adenovirus thatcould highly overexpress the tumor suppressor DBC2 gene in a bladder cancer cell line. This viral construct would be widelyused for our further research in gene functional assays and gene therapy in bladder cancer.