SignificanceWe report on a zebrafish model of hearing loss in Usher syndrome type IIIA caused by pathogenic variant c.144T>G (p.N48K) in the clarin1 gene (CLRN1). We expressedCLRN1c.144T>Gin wild-type or clarin1 knockout zebrafish and then probed these animals with genetic or pharmaceutical small-molecular agents. Our findings revealed that the CLRN1N48Kprotein is transported to the hair cell bundle via an unconventional secretory pathway, and activation of this pathway using artemisinin, an antimalarial drug, enhanced CLRN1N48Klevels in hair cell bundles and rescued their functions. Our report demonstrates a paradigm to evaluate the functional consequence of human pathogenic variants in hair cells in vivo and reveals artemisinin’s therapeutic potential to mitigate sensory deficits inCLRN1c.144T>Gand other endoplasmic reticulum aggregation disorders.