Transient receptor potential vanilloid (TRPV) 4 is a nonselective cation channel expressed in sensory neurons such as those in the dorsal root and trigeminal ganglia, kidney, and inner ear. TRPV4 is activated by mechanical stress, heat, low osmotic pressure, low pH, and phorbol derivatives such as 4α-phorbol 12,13-didecanoate (4α-PDD). We investigated the expression of TRPV4 in rat vestibular ganglion (VG) neurons. The TRPV4 gene was successfully amplified from VG neuron mRNA using reverse-transcription polymerase chain reaction. Furthermore, immunoblotting showed positive expression of TRPV4 protein in VG neurons. Immunohistochemistry indicated that TRPV4 was localized predominantly on the plasma membrane of VG neurons. Calcium (Ca2+) imaging of VG neurons showed that 4α-PDD and/or hypotonic stimuli caused an increase in intracellular Ca2+ concentration ([Ca2+]i) that was almost completely inhibited by ruthenium red, a selective antagonist of TRPV channels. Interestingly, a [Ca2+]i increase was evoked by both hypotonic stimuli and 4α-PDD in approximately 38% of VG neurons. These data indicate that TRPV4 is functionally expressed in VG neurons as an ion channel and that TRPV4 likely participates in VG neurons for vestibular neurotransmission as an osmoreceptor and/or mechanoreceptor.