Mutations inGJB2(Gap junction protein beta 2) are the most common genetic cause of non-syndromic hereditary deafness in humans, especially the 35delG and 235delC mutations. Owing to the homozygous lethality ofGjb2mutations in mice, there are currently no perfect mouse models carryingGjb2mutations derived from patients for mimicking human hereditary deafness and for unveiling the pathogenesis of the disease. Here, we successfully constructed heterozygousGjb2+/35delGandGjb2+/235delCmutant mice through advanced androgenic haploid embryonic stem cell (AG-haESC)-mediated semi-cloning technology, and these mice showed normal hearing at postnatal day (P) 28. A homozygous mutant mouse model,Gjb235delG/35delG, was then generated using enhanced tetraploid embryo complementation, demonstrating that GJB2 plays an indispensable role in mouse placenta development. These mice exhibited profound hearing loss similar to human patients at P14, i.e., soon after the onset of hearing. Mechanistic analyses showed thatGjb235delG disrupts the function and formation of intercellular gap junction channels of the cochlea rather than affecting the survival and function of hair cells. Collectively, our study provides ideal mouse models for understanding the pathogenic mechanism of DFNB1A-related hereditary deafness and opens up a new avenue for investigating the treatment of this disease.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-023-04794-9.