Background Bioreactors are pivotal tools for generating mechanical stimulation in functional tissue engineering study.This study aimed to create a bioreactor that can simulate urinary bladder mechanical properties, and to investigate the effects of a mechanically stimulated culture on urothelial cells and bladder smooth muscle cells.Methods We designed a bioreactor to simulate the mechanical properties of bladder. A pressure-record system was used to evaluate the mechanical properties of the bioreactor by measuring the pressure in culture chambers. To test the biocompatibility of the bioreactor, viabilities of urothelial cells and smooth muscle cells cultured in the bioreactor under static and mechanically changed conditions were measured after 7-day culture. To evaluate the effect of mechanical stimulations on the vital cells, urethral cells and smooth muscle cells were cultured in the simulated mechanical conditions. After that, the viability and the distribution pattern of the cells were observed and compared with cells cultured in non-mechanical stimulated condition.Results The bioreactor system successfully generated waveforms similar to the intended programmed model while maintaining a cell-seeded elastic membrane between the chambers. There were no differences between viabilities of urothelial cells ((91.90±1.22)% vs. (93.14±1.78)%, P ＞0.05) and bladder smooth muscle cells ((93.41±1.49)% vs.(92.61±1.34)%, P ＞0.05). The viability of cells and tissue structure observation after cultured in simulated condition showed that mechanical stimulation was the only factor affected cells in the bioreactor and improved the arrangement of cells on silastic membrane.Conclusions This bioreactor can effectively simulate the physiological and mechanical properties of the bladder.Mechanical stimulation is the only factor that affected the viability of cells cultured in the bioreactor. The bioreactor can change the growth behavior of urothelial cells and bladder smooth muscle cells, resulting in the cells undergoing adaptive changes in mechanically-stimulated environment.