This chapter reviews the use of urothelial cells as a means to enhance tissue regeneration and wound healing in urinary tract system. It addresses the properties of urothelial cells, including their role as a permeability barrier to protect underlying muscle tissue from the caustic effects of urine and as one of the main cell types, along with smooth muscle cells, that are used in urethral or bladder tissue engineering today. This description includes a general overview of various isolation techniques and culture methods that have been developed to improve urinary tract reconstruction in vivo and aid the characterization of growth factor expression in vitro. The chapter then describes various applications using urothelial cells, including production of multilayer urothelial sheets, tissue engineered bladder mucosa, tissue engineered urethra, and tissue engineered bladder. It also outlines the advantages of sandwich and layered coculture of these cells and the effects of epithelial-stromal cell interactions during tissue regeneration or wound healing processes in the urinary tract.