Background: ATOH1 is recognized as a crucial transcription factor for hair cell differentiation. Although Atoh1 expression has been documented in a subset of developing spiral ganglion neurons, its specific role in neuronal development remains to be elucidated.Methods: We generated a novel conditional deletion model (Atoh1Cre-Isl1CKO) targeting Isl1 in the inner ear. Immunohistochemistry was used to examine morphological and cellular changes in the inner ear. Additionally, we assessed the hearing function of Atoh1Cre-Isl1CKO mutants compared to littermate control mice using auditory brainstem responses (ABRs) and distortion product otoacoustic emission (DPOAE) tests.Results: The conditional deletion of Isl1 did not impact the survival of Atoh1Cre-Isl1CKO mutants. The vestibular organs appeared normal in size, with hair cells and innervation indistinguishable from those of control littermates. Similarly, neither the hair cells nor supporting cells in the cochlea were affected. However, the spiral ganglion in the apex exhibited a significant reduction in neurons, resulting in a less compact structure with neurons more widely dispersed and aberrant innervation observed at P0. The morphological changes were associated with significant hearing impairment in adult mice. In comparison with controls, the DPOAEs, but especially ABRs in Atoh1Cre-Isl1CKO were significantly affected in low and middle frequencies.Conclusions: We demonstrated that altering the molecular properties of these neurons through the elimination of Isl1 resulted in disrupted spiral ganglion development. Consequently, the loss of these neurons had a detrimental effect on the hearing function of Atoh1Cre-Isl1CKO mutants. Supported by the Czech Science Foundation (GA23-05963S).