Objective: To assess the oncolytic effect of fiber-substituted conditionally replicating adenovirus type 5 (Ad5) F35 vector on human bladder cancer cell lines such as 253J, 5637, KK-47, T24, TCCSUP, and UMUC-3 cells. Materials and Methods: Ad5F35 and Ad5 conditionally replicating adenovirus vectors containing the E1 gene controlled by the human midkine promoter (Ad5F35/MKp-E1 and Ad5/MKp-E1, respectively) were constructed. Reverse transcriptase-polymerase chain reaction and cell viability assay were performed in cells transfected with Ad5F35/MKp-E1 or Ad5/MKp-E1. Results: Of the bladder cancer cells used, considerably lower expression of mRNA for Coxsackie and adenovirus receptor, an Ad5 receptor, was found with T24 and TCCSUP cells. However, the mRNA for CD46, an Ad35 receptor, was abundantly expressed in all the cell types. Ad5F35/MKp-E1 induced oncolysis in a plaque formation unit-dependent manner for all the bladder cancer cells used, with greater efficacy than Ad5/MKp-E1 for T24, TCCSUP, and 253J cells. Conclusion: The results of the present study have shown that Ad5F35/MKp-E1 is more useful for the gene therapy of bladder cancer than Ad5/MKp-E1 is for some cell lines.