Proper mammalian hearing depends on an outer hair cell-based mechanism that amplifies the sound-induced travelling waves in the cochlea. Outer hair cells (OHCs) contribute to this cochlear amplification through their electromotile property—voltage-dependent somatic length changes that can operate at acoustic frequencies. This unique form of motility is driven by prestin, a member of the solute carrier 26 family of anion transporters that is highly expressed along the OHC lateral plasma membrane. The lateral plasma membrane is supported by a cortical actin-spectrin lattice and a smooth ER system known as lateral cisternae to form a regular layered structure along the entire OHC lateral wall. The detailed structural organization of each layer and how they interact to transduce prestin conformational changes into whole-cell motility are not well understood. In this dissertation, I combine cryogenic sample preparation methods and electron tomography to elucidate the functional architecture of the OHC lateral wall complex. In chapter 1, I review the biology of the mammalian auditory system. In chapter 2, I detail how the combined methodological approach used can preserve and reveal the three-dimensional nano-architectures in cells at near-native state. In chapter 3, I describe the successful use of this methodology to elucidate the structure-function relationships in a comparable model structure, the glycocalyx on the surface of enterocytes. In Chapter 4, I provide the details on the organization of each layer of the OHC lateral wall complex and how they are structurally integrated. I show that the lateral plasma membrane contains closely tiled microdomains of orthogonally packed putative prestin protein complexes. The cortical lattice connects the plasma membrane to the adjacent lateral cisternae through two independent cross-bridging components. The lateral cisternae are in turn integrated through inter and intra-cisternal cross-bridging systems. Finally, mitochondria are attached to the lateral cisternae through another set of linker elements. By quantifying the dimensions of each of these components and mapping their distribution I provide a detailed blueprint of the nano-architecture of the OHC electromotile apparatus and discuss how its cohesive structure allows effective transmission of forces generated by prestin to the rest of the cell to drive cochlear amplification.