Transient receptor potential vanilloid 4 (TRPV4), a cation channel responsive to hypotonicity, can also be activated by warm temperatures. Moreover, TRPV4^-/- mice reportedly exhibit deficits in inflammation-induced thermal hyperalgesia. However, it is unknown whether TRPV4 or related transient receptor potential channels account for warmth perception under injury-free conditions. We therefore investigated the contribution of TRPV4 to thermosensation and thermoregulation in vivo. On a thermal gradient, TRPV4^-/- mice selected warmer floor temperatures than wild-type littermates. In addition, whereas wild-type mice failed to discriminate between floor temperatures of 30 and 34°C, TRPV4^-/- mice exhibited a strong preference for 34°C. TRPV4^-/- mice also exhibited prolonged withdrawal latencies during acute tail heating. TRPV4^-/- and wild-type mice exhibited similar changes in behavior on a thermal gradient after paw inflammation. Circadian body temperature fluctuations and thermoregulation in a warm environment were also indistinguishable between genotypes. These results demonstrate that TRPV4 is required for normal thermal responsiveness in vivo.