Significance: We provide the first direct evidence of the urothelial response to water transport through the urothelium, traditionally considered impermeable. Using optical coherence tomography (OCT), we observe that the urothelium absorbs and expels water under varying concentrations of NaCl, challenging long-held views about its impermeability. The discovery that osmotic stress can induce urothelial damage has implications for bladder disorders such as interstitial cystitis and overactive bladder, where urothelial integrity is compromised. Aim: Traditionally considered impermeable, the urothelium has recently been implicated in water transport due to the presence of aquaporins. Despite this, direct evidence of the urothelial response to water movement through the urothelium remains elusive. We aim to provide such evidence by examining urothelial responses to NaCl solutions using OCT. Approach: Fresh porcine bladder samples were subjected to OCT imaging to observe urothelial responses under varying osmolarity conditions, using NaCl solutions ranging from 0.31 to Results: OCT imaging revealed a significant increase in urothelial optical pathlength thickness following deionized water application, indicative of water absorption. Conversely, exposure to higher osmolarity NaCl solutions resulted in urothelial shrinkage, suggesting water efflux. Histological analysis demonstrated intact cellular structures at lower osmolarities ( Conclusions: We provide evidence that the urothelium is a dynamic barrier capable of water transport, influenced by osmotic gradients. The observed osmotic-induced urothelial damage may have important implications for the pathophysiology of conditions such as interstitial cystitis and overactive bladder, offering insights into potential diagnostic and therapeutic strategies. These findings warrant further investigation using human tissue.