Physiological brain temperature is an important determinant of brain functions, and it is well established that changes in brain temperature dynamically influence hippocampal neuronal activity. We previously revealed that the thermo-sensor TRPV4 (activated above 34°C) is activated by physiological brain temperature in hippocampal neurons and thereby controls their excitability in vitro (J. Neurosci. 2007, Shibasaki et al.). Here, we examined whether TRPV4 regulates neuronal excitability through its activation by brain temperature in vivo. We developed an original device to cool local brain temperature to inactivate TRPV4. The cooling treatment clearly demonstrated that constitutive TRPV4 activation was occurred in mouse brain, and we found that hippocampal theta-frequency electroencephalogram (EEG) activities in TRPV4KO mice during wake periods were significantly reduced compared with those in WT mice. Furthermore, slice patch clamp recordings from dentate gyrus of hippocampus revealed that WT neurons further depolarized compared with TRPV4KO neurons. Depending on the differences of the resting membrane potentials, WT neurons had smaller fEPSPs and higher firing than KO neurons at 35°C (above TRPV4 activation), however, the differences were abolished at 30°C (less than TRPV4 activation). Taken together, for the first time we reveal that TRPV4 is an important transducer that converts brain temperature into neuronal electrical excitability in mammals.