The sensitivity and frequency selectivity of the mammalian cochlea involves a mechanical amplification process called electromotility, which requires prestin-dependent length changes of the outer hair cell (OHC) lateral wall in response to changes in membrane electric potential. The cortical lattice, the highly organized cytoskeleton underlying the OHC lateral plasma membrane, is made up of F-actin and spectrin. Here, we show that alphaII and two of the five beta-spectrin subunits, betaII and betaV, are present in OHCs. betaII spectrin is restricted to the cuticular plate, a dense apical network of actin filaments, whereas betaV spectrin is concentrated at the cortical lattice. Moreover, we show that alphaII-betaV spectrin directly interacts with F-actin and band 4.1, two components of the OHC cortical lattice. betaV spectrin is progressively recruited into the cortical lattice between postnatal day 2 (P2) and P10 in the mouse, in parallel with prestin membrane insertion, which itself parallels the maturation of cell electromotility. Although betaV spectrin does not directly interact with prestin, we found that addition of lysates derived from mature auditory organs, but not from the brain or liver, enables betaV spectrin-prestin interaction. Using this assay, betaV spectrin, via its PH domain, indirectly interacts with the C-terminal cytodomain of prestin. We conclude that the cortical network involved in the sound-induced electromotility of OHCs contains alphaII-betaV spectrin, and not the conventional alphaII-betaII spectrin.