Unbiased genetic screens implicated a number of uncharacterized genes in hearing loss, suggesting some biological processes required for auditory function remain unexplored. Loss of /, a previously understudied gene, caused deafness in mice, but how it functioned in the hearing was unclear. Here, we show that disruption of causes hearing loss in the zebrafish. Defects in mechanotransduction, longer and thinner hair bundles, and enlarged apical lysosomes in hair cells are observed in the mutant. In cultured cells, Kiaa1024L/Minar2 is mainly localized to lysosomes, and its overexpression recruits cholesterol and increases cholesterol labeling. Strikingly, cholesterol is highly enriched in the hair bundle membrane, and loss of reduces cholesterol localization to the hair bundles. Lowering cholesterol levels aggravates, while increasing cholesterol levels rescues the hair cell defects in the mutant. Therefore, cholesterol plays an essential role in hair bundles, and Kiaa1024L/Minar2 regulates cholesterol distribution and homeostasis to ensure normal hearing.