Water-intake behavior is under the control of brain systems that sense body-fluid conditions at sensory circumventricular organs (sCVOs); however, the underlying mechanisms have not yet been elucidated in detail. Na_x is a sodium (Na⁺) level sensor in the brain, and the transient receptor potential vanilloid (TRPV) channels, TRPV1 and TRPV4, have been proposed to function as osmosensors. We herein investigated voluntary water intake immediately induced after an intracerebroventricular (icv) administration of a hypertonic NaCl solution in TRPV1-, TRPV4-, Na_x-, and their double-gene knockout (KO) mice. The induction of water intake by TRPV1-KO mice was normal, whereas that by TRPV4-KO and Na_x-KO mice was significantly less than that by WT mice. Water intake by Na_x/TRPV4-double KO mice was similar to that by the respective single KO mice. When TRPV4 activity was blocked with a specific antagonist HC-067047, water intake by WT mice was significantly reduced, whereas that by TRPV4-KO and Na_x-KO mice was not. Similar results were obtained with the administration of miconazole, which inhibits the biosynthesis of epoxyeicosatrienoic acids (EETs), endogenous agonists for TRPV4, from arachidonic acid (AA). Icv injection of hypertonic NaCl with AA or 5,6-EET restored water intake by Na_x-KO mice to the WT level, but not that by TRPV4-KO mice. These results suggest that the Na⁺ signal generated in Na_x-positive glial cells leads to the activation of TRPV4-positive neurons in sCVOs in order to stimulate water intake by using EETs as gliotransmitters.