Hydrogen peroxide (H₂O₂) has important messenger and effector functions in the plant and animal kingdom. Phagocytes produce H₂O₂ to kill pathogens, and epithelial cells of large airways have also been reported to produce H₂O₂ for signaling and host defense purposes. In this report, we show for the first time that urothelial cells produce H₂O₂ in response to a calcium signal. Using a gene-deficient mouse model we also demonstrate that H₂O₂ is produced by the NADPH oxidase Duox1, which is expressed in the mouse urothelium. In contrast, we found no evidence for the expression of lactoperoxidase, an enzyme that has been shown to cooperate with Duox enzymes. We also found that specific activation of TRPV4 calcium channels elicits a calcium signal and stimulates H₂O₂ production in urothelial cells. Furthermore, we detected altered pressure responses in the urinary bladders of Duox1 knockout animals. Our results raise the possibility that mechanosensing in epithelial cells involves calcium-dependent H₂O₂ production similar to that observed in plants.