Osmoreception is essential for systemic osmoregulation, a process to stabilize the tonicity and volume of the extracellular fluid through regulating the ingestive behaviour, sympathetic outflow and renal function. The sensation of osmotic changes by osmoreceptor neurons is mediated by ion channels that detect the change of osmolarity in extracellular fluid. However, the molecular identity of these channels remains mysterious. AtCSC1and OSCA1, two closely related paralogues from Arabidopsis, have been demonstrated to form hyperosmolarity activated ion channels, which makes their mammalian orthologues-the members of TMEM63 proteins, possible candidates for osmoreceptor transduction channel. To test this possibility, we cloned the cDNAs of all the three members of the mouse TMEM63 family, TMEM63A, TMEM63B and TMEM63C from the mRNA from mouse brain. When all of the three subtypes of TMEM63 proteins were co-expressed in HEK293 cells, we recorded membrane currents evoked by hypertonic stimulation in these cells. However, the cells expressing the combinations of any two subtypes of TMEM63 proteins could not exhibit any hyperosmolarity evoked currents. Thus, all the three members of TMEM63 proteins are required to constitute a hyperosmolarity activated ion channel. We propose that the TMEM63 proteins may serve as an osmolarity sensitive ion channel for the osmoreception. Copyright (C) 2016 John Wiley & Sons, Ltd.