Protocadherin‐15 (Pcdh15) is a component of the tip‐links, the extracellular filaments that gate hair cell mechano‐electrical transduction channels in the inner ear. There are three Pcdh15 splice isoforms (CD1, CD2 and CD3), which only differ by their cytoplasmic domains; they are thought to function redundantly in mechano‐electrical transduction during hair‐bundle development, but whether any of these isoforms composes the tip‐link in mature hair cells remains unknown. By immunolabelling and both morphological and electrophysiological analyses of post‐natal hair cell‐specific conditional knockout mice (Pcdh15^{ex38‐fl/ex38‐fl} Myo15‐cre^+/−) that lose only this isoform after normal hair‐bundle development, we show that Pcdh15‐CD2 is an essential component of tip‐links in mature auditory hair cells. The finding, in the homozygous or compound heterozygous state, of a PCDH15 frameshift mutation (p.P1515Tfs*4) that affects only Pcdh15‐CD2, in profoundly deaf children from two unrelated families, extends this conclusion to humans. These results provide key information for identification of new components of the mature auditory mechano‐electrical transduction machinery. This will also serve as a basis for the development of gene therapy for deafness caused by PCDH15 defects.