We have developed a simple multi-layer microfluidic device by integrating a polydimethyl siloxane (PDMS) microfluidic channel and a porous membrane substrate to culture and analyze the renal tubular cells. As a model cell type, primary rat inner medullary collecting duct (IMCD) cells were cultured inside the channel. To generate in vivo-like tubular environments for the cells, a fluidic shear stress of 1 dyn/cm(2) was applied for 5 hours, allowing for optimal fluidic conditions for the cultured cells, as verified by enhanced cell polarization, cytoskeletal reorganization, and molecular transport by hormonal stimulations. These results suggest that the microfluidic device presented here is useful for resembling an in vivo renal tubule system and has potential applications in drug screening and advanced tissue engineering.