CasRx, a recently discovered member of the type VI CRISPR system with minimum size, offers a new approach for RNA manipulation with high efficiency and specificity in prokaryotes and eukaryotes. However, studies of functional recovery using the CasRx system have not been well characterized. Here, we sought to establish an adeno-associated virus (AAV)-CasRx-guide RNA (gRNA) system for the specific knockdown of transcript to protect mice from aminoglycosides-induced hearing loss. For the study, we verified an optimized gRNA , which was packaged into a single AAV with CasRx, and injected the packaged AAV into mice with hearing loss induced by neomycin and auditory functions investigated by auditory brainstem response tests. Upon using the AAV-CasRx-gRNA system, we found the knockdown of transcript led to less cochlear hair cell loss and improved auditory function, with low off-target and adverse side effects. Additionally, the decrease in significantly inhibits mRNA expression of and . In conclusion, the AAV-CasRx-gRNA-mediated knockdown of transcript in mice has been proved effective and safe for preventing hearing loss induced by aminoglycosides and, thus, represents a promising genetic approach for the future clinical applications for treating non-inherited hearing loss.