Auditory detection is essential for survival and reproduction of vertebrates, yet the genetic changes underlying the evolution and diversity of hearing are poorly documented. Recent discoveries concerning prestin, which is responsible for cochlear amplification by electromotility, provide an opportunity to redress this situation. We identify prestin genes from the genomes of 14 vertebrates, including three fishes, one amphibian, one lizard, one bird, and eight mammals. An evolutionary analysis of these sequences and 34 previously known prestin genes reveals for the first time that this hearing gene was under positive selection in the most recent common ancestor (MRCA) of tetrapods. This discovery might document the genetic basis of enhanced high sound sensibility in tetrapods. An investigation of the adaptive gain and evolution of electromotility, an important evolutionary innovation for the highest hearing ability of mammals, detects evidence for positive selections on the MRCA of mammals, therians, and placentals, respectively. It is suggested that electromotility determined by prestin might initially appear in the MRCA of mammals, and its functional improvements might occur in the MRCA of therian and placental mammals. Our patch clamp experiments further support this hypothesis, revealing the functional divergence of voltage-dependent nonlinear capacitance of prestin from platypus, opossum, and gerbil. Moreover, structure-based cdocking analyses detect positively selected amino acids in the MRCA of placental mammals that are key residues in sulfate anion transport. This study provides new insights into the adaptation and functional diversity of hearing sensitivity in vertebrates by evolutionary and functional analysis of the hearing gene prestin.