Murine models describe a defined host/pathogen interaction for urinary tract infection, but human cell studies are scant. Although recent human urothelial organoid models are promising, none demonstrate long-term tolerance to urine, the natural substrate of the tissue and of the uropathogens that live there. We developed a novel human organoid from progenitor cells which demonstrates key structural hallmarks and biomarkers of the urothelium. After three weeks of transwell culture with 100% urine at the apical interface, the organoid stratified into multiple layers. The apical surface differentiated into enlarged and flattened umbrella-like cells bearing characteristic tight junctions, structures resembling asymmetric unit membrane plaques, and a glycosaminoglycan layer. The apical cells also expressed cytokeratin-20, a spatial feature of the mammalian urothelium. Urine itself was necessary for full development, and undifferentiated cells were urine-tolerant despite the lack of membrane plaques and a glycosaminoglycan layer. Infection with Enterococcus faecalis revealed the expected invasive outcome, including urothelial sloughing and the formation of intracellular colonies similar to those previously observed in patient cells. This new biomimetic model could help illuminate invasive behaviours of uropathogens, and serve as a reproducible test bed for disease formation, treatment and resolution in patients.