Hereditary deafness affects about 1 in 2000 children and mutations in the GJB2 gene are the major cause in various ethnic groups. GJB2 encodes connexin26, a putative channel component in cochlear gap junction. However, the pathogenesis of hearing loss caused by the GJB2 mutations remains obscure. The generation of a mouse model to study the function of connexin26 during hearing has been hampered by the fact that Gjb2 knockout mice are embryonic lethal. To establish viable model mice we generated transgenic mice expressing a mutant connexin26 with R75W mutation that was identified in a deaf family with autosomal-dominant inheritance. The previous expression analysis revealed that the R75W connexin26 inhibited the gap channel function of the co-expressed normal connexin26 in a dominant-negative fashion. We established two lines of transgenic mice that showed severe to profound hearing loss, deformity of supporting cells, failure in the formation of the tunnel of Corti and degeneration of sensory hair cells. Despite robust expression of the transgene, no obvious structural change was observed in the stria vascularis or spiral ligament that is rich in connexin26 and generates the endolymph. The high resting potential in cochlear endolymph essential for hair cell excitation was normally sustained. These results suggest that the GJB2 mutation disturbs homeostasis of cortilymph, an extracellular space surrounding the sensory hair cells, due to impaired K⁺ transport by supporting cells, resulting in degradation of the organ of Corti, rather than affecting endolymph homeostasis in mice and probably in humans.