Significance The mammalian auditory organ harbors a subpopulation of sensory hair cells that alternately shorten and elongate in response to sound stimuli. This singular feature, called somatic electromotility, is associated with the presence of a flexible cortical network of F-actin and spectrin βV, a large unconventional β-spectrin. We show that adaptive mutations of spectrin-βV occurred in the mammalian lineage and were accompanied with substantial changes in the protein distribution within inner ear hair cells. This study illustrates how a comparison of the subcellular locations of a protein between nonmammalian and mammalian vertebrates in a given cell type, combined with branch-site model analysis of the protein family in silico, can shed light on a major mammalian structural/functional innovation.