Choroid plexus is involved in the maintenance of the brain’s environment in the lateral, the third and the forth ventricles. This tissue consists of a continuous monolayer structure made of epithelial cells, leptomeninges and fenestrated capillaries. Choroid plexus epithelial cells (CPECs) are separated into apical and basolateral sides by tight junctions. The apical membrane faces the cerebrospinal fluid (CSF) in the ventricles whereas the basolateral membrane is on the side of the fenestrated capillaries. This structure is very important for formation of the blood-CSF barrier, and the most important function of CPECs is CSF production. The generation of CSF is dependent upon electrolyte transport from the basolateral to the apical membranes of CPECs and involves many ion transport proteins. On the other hand, TRPV4, a calcium-permeable channel, is highly expressed in the apical membrane of CPECs in the brain while its physiological function is poorly understood. We observed outwardly rectifying chloride currents induced by intracellular calcium increases in isolated murine CPECs although the functional expression of calcium-activated chloride channels (CaCCs) had not been reported. Therefore, we hypothesized that TRPV4 activation might affect CaCC activity. We found co-expression of anoctamin 1 (ANO1), one of CaCCs, with TRPV4 in CPECs. Therefore, physical and functional interaction between TRPV4 and ANO1 was examined in HEK293T cells and CPECs (1). Chloride currents induced by a TRPV4 activator GSK1016790A (GSK) were markedly increased in an extracellular calcium-dependent manner in HEK293T cells expressing TRPV4 with ANO1, but not with ANO4, ANO6 or ANO10, the mRNAs of which were expressed in the choroid plexus. We also found physical interaction between TRPV4 and ANO1 in both HEK293T cells and choroid plexus. We observed that ANO1 was activated at a warm temperature (37 degree C) in HEK293T cells and that the heat-evoked chloride currents were markedly enhanced after GSK application in CPECs. Simultaneous stimulation by warmth and hyposmosis induced chloride current activation in wild-type, but not in TRPV4-deficient CPECs. Cell volume changes were induced by ANO1-mediated chloride currents in parallel with membrane potential changes, and the cell volume was significantly decreased at negative membrane potentials by TRPV4-induced ANO1 activation. In native cells, cation efflux should accompany chloride movement and could help to maintain the deep membrane potentials that in turn accelerate chloride efflux. Thus, physical and functional interactions between TRPV4 and ANO1 can modulate water transport in the choroid plexus. The functional interaction between TRPV4 and ANO1 was also observed in the exocrine glands with water efflux, and the functional interaction between TRP channels with high calcium permeability and ANO1 is widely observed in other cell types such as sensory neurons.