The urinary tract is exposed to a variety of possible injures that may lead to organ damage or loss, and thus, the establishment of valid in vitro urothelial models to study the mechanism of drug candidates is necessary. This study is the first to investigate the effect of chitosan on urothelia in vitro and to evaluate whether chitosan-treated urothelial models can regenerate in vitro and reestablish a functional urothelium. Biomimetic hyperplastic and normoplastic urothelial models were used to test the effect of chitosan (0.05%) on partially and highly differentiated urothelial cells (UCs) by monitoring their molecular, ultrastructural, and physiological changes for 3 weeks. Chitosan caused an immediate and complete loss of transepithelial resistance (TER), tight junction disruption, cytopathological changes of UCs, and consequently enhanced the permeability of partially and highly differentiated urothelial models. However, 3 weeks after chitosan treatment, TER was reestablished, tight junctions resealed, permeability decreased, and progressive differentiation stages of newly exposed superficial UCs expressing uroplakins and tight junction protein claudin-8 were found. The in vitro models regenerated and reestablished urothelia with a tight barrier. The biomimetic urothelial models represent appropriate in vitro models for studying urothelial drug candidates as well as evaluating drug permeabilities and their intracellular function. Understanding the possible intracellular function of chitosan could significantly advance approaches to treating urothelial-specific diseases.