Balance dysfunction is closely associated with loss of vestibular hair cells (HCs). Gene therapy shows promise when used to protect or regenerate vestibular HCs to preserve or restore adequate vestibular function. Adeno-associated virus (AAV) vectors allow long-term gene expression in the absence of toxicity. To noninvasively define an AAV serotype exhibiting favorable tropism toward the vestibular sensory epithelium, we characterized the transgene expression potential of AAV vectors (serotypes 1, 2, 5, 6 and 8) inoculated into adult mouse utricle via canalostomy. We found that AAV8 was the most effective AAV vector in utricular gene transfer. Swim tests and measurements of auditory brainstem response revealed minimal loss of vestibular function and hearing after canalostomy. In the normal utricle after AAV8 infusion, transduction efficiency peaked at 7 days, and was maintained thereafter, in vestibular HCs, and at 3 days in supporting cells (SCs). In the streptomycin-lesioned utricle, the SC transduction efficiency peaked at 7 days and decreased at 30 days. In conclusion, AAV8-mediated gene transfer via canalostomy facilitates efficient and safe transduction in mouse vestibular sensory epithelium, and may in the future become clinically relevant for human vestibular gene therapy.