Urine is a dynamic and highly variable biofluid. Urine-urothelium interactions are a critical yet underexplored factor in bladder homeostasis and urinary tract infections (UTIs). Here, we report on a human ‘mini-bladder’ model that exposes a stratified urothelium to urine of defined composition, and incorporates micturition. Prolonged exposure to high-solute concentration urine weakens tight junctions, dysregulates immune responses, and reduces bladder tissue resilience. This increases susceptibility to colonization of the bladder by uropathogenic E. coli (UPEC) which reduces efficacy of antibiotic therapy. In high-solute concentration urine, Fosfomycin monotherapy – prescribed for uncomplicated UTIs, induces the formation of cell wall-deficient (CWD) UPEC in the urine (as observed in patients with recurrent UTIs) but also within deeper urothelial layers. Tissue-associated CWD UPEC directly contributed to recurrence. Our findings expand the conceptual role for CWD UPEC in UTIs, and demonstrate the power of the mini-bladder platform to capture urine-urothelial microenvironment dynamics that actively shape UTI pathogenesis and antibiotic tolerance.