All mucosal surfaces must deal with the challenge of exposure to the outside world. The urothelium is a highly specialized layer of stratified epithelial cells lining the inner surface of the urinary bladder, a gruelling environment involving significant stretch forces, osmotic and hydrostatic pressures, toxic substances, and microbial invasion. The urinary bladder plays an important barrier role and allows the accommodation and expulsion of large volumes of urine without permitting urine components to diffuse across. The urothelium is made up of three cell types, basal, intermediate, and umbrella cells, whose specialized functions aid in the bladder's mission. In this review, we summarize the recent insights into urothelial structure, function, development, regeneration, and in particular the role of umbrella cells in barrier formation and maintenance. We briefly review diseases which involve the bladder and discuss current human urothelial in vitro models as a complement to traditional animal studies.