The V-shaped arrangement of hair bundles on cochlear hair cells is critical for the auditory sensing. However, regulation of hair bundle arrangements is not fully understood. Recently, defects in hair bundle arrangement were reported in postnatal Dishevelled-associating protein (Daple)-deficient mice. Here, we found that adult Daple-/- mice exhibited hearing disturbances over a broad frequency range through auditory brainstem response testing. Consistently, distorted patterns of hair bundles were detected in almost all regions, more typically in the basal region of the cochlear duct. In adult Daple-/- mice, apical microtubules were irregularly aggregated, and the number of microtubules attached to plasma membranes was decreased. Similar phenotypes were manifested upon nocodazole treatment in a wild type cochlea culture without affecting the microtubule structure of the kinocilium. These results indicate critical roles of Daple in hair bundle arrangement, through the orchestration of apical microtubule distribution, and thereby in hearing, especially at high frequencies.