Transient receptor potential channel vanilloid subfamily 4 (TRPV4), a member of TRP family, is a mechanosensitive non-selective cation channel. To investigate the role of TRPV4 in the cochlea, the hearing thresholds and effects of acoustic overexposure on the cochlea were examined in TRPV4 knockout mice. TRPV4 knockout mice at age 8 weeks exhibited normal, but those at 24 weeks revealed significantly higher thresholds by auditory brainstem response. The auditory threshold shift was significantly larger in the TRPV4 knockout than in the TRPV4+/+ mice 1 week after the acoustic overexposure of 128dB SPL. The present findings suggest that disruption of TRPV4 causes delayed-onset hearing loss and makes the cochlea vulnerable to acoustic injury.