Here we provide evidence for a critical role of the transient receptor potential cation channel, subfamily V, member 4 (TRPV4) in normal bladder function. Immunofluorescence demonstrated TRPV4 expression in mouse and rat urothelium and vascular endothelium, but not in other cell types of the bladder. Intracellular Ca 2+ measurements on urothelial cells isolated from mice revealed a TRPV4-dependent response to the selective TRPV4 agonist 4α-phorbol 12, 13-didecanoate and to hypotonic cell swelling. Behavioral studies demonstrated that TRPV4–/–mice manifest an incontinent phenotype but show normal exploratory activity and anxiety-related behavior. Cystometric experiments revealed that TRPV4–/–mice exhibit a lower frequency of voiding contractions as well as a higher frequency of nonvoiding contractions. Additionally, the amplitude of the spontaneous contractions in explanted bladder strips from TRPV4–/–mice was significantly reduced. Finally, a decreased intravesical stretch-evoked ATP release was found in isolated whole bladders from TRPV4–/–mice. These data demonstrate a previously unrecognized role for TRPV4 in voiding behavior, raising the possibility that TRPV4 plays a critical role in urothelium-mediated transduction of intravesical mechanical pressure.