Urothelial cells, the cells lining the surface of the urinary bladder, comprise of a unique cell type with high plasticity and a variety of cell functions. They are the first line of bladder defense and serve as an interface between pathogens. Urothelial cells have at their disposal several defense mechanisms to prevent adherence of pathogens and maintain impermeability to urinary solutes. Urothelial cells express both estrogen receptor-alpha and beta, epidermal growth factor receptor and fibroblast growth factor receptor. These receptors play a major role in urothelial cell response to injury and infection. The urothelial cells also release a number of cytokines and other immune system mediators. The ability to culture normal urothelial cells in vitro make it possible to further investigate the immunoregulatory potential of urothelial cells. Human urothelial cells are also useful tools to develop in vitro disease models for High Throughput and High Content Screening.