The state of the skin changes drastically depending on the ambient temperature. Skin epidermal keratinocytes express thermosensitive transient receptor potential vanilloid (TRPV) cation channels, TRPV3 and TRPV4. These multimodal receptors are activated by various kinds of chemical and physical stimuli, including warm temperatures (>30°C). It has been suggested that TRPV4 is involved in cell-cell junction maturation; however, the effect of temperature fluctuations on TRPV4-dependent barrier homeostasis is unclear. In the present study, we demonstrated that activation of TRPV4 was crucial for barrier formation and recovery, both of which were critical for the prevention of excess dehydration of human skin keratinocytes. TRPV4 activation by physiological skin temperature (33°C), GSK1016790A or 4α-PDD allowed influx of Ca(2+) from extracellular spaces which promoted cell-cell junction development. These changes resulted in augmentation of intercellular barrier integrity in vitro and ex vivo. TRPV4 disruption reduced the increase in trans-epidermal resistance and increased intercellular permeation after a Ca(2+) switch. Furthermore, barrier recovery after the disruption of the stratum corneum was accelerated by the activation of TRPV4 either by warm temperature or a chemical activator. Our results suggest that physiological skin temperatures play important roles in cell-cell junction and skin barrier homeostasis through TRPV4 activation.