Auditory hair cell regeneration following injury is critical to hearing restoration. The Notch signaling pathway participates in the regulation of inner ear development and cell differentiation. Recent evidence suggests that microRNA (miR)‑183 has a similar role in the inner ear. However, it is unclear how Notch signaling functions in hair cell regeneration in mammals and if there is cross‑talk between Notch signaling and miR‑183. The present study used a gentamicin‑induced cochlear injury mouse model. Gentamicin‑induced damage of the hair cells activated the Notch signaling pathway and downregulated miR‑183 expression. Notch signaling inhibition by the γ‑secretase inhibitor, 24‑diamino‑5‑phenylthiazole (DAPT), attenuated gentamicin‑induced hair cell loss and reversed the downregulation of miR‑183 expression. Further investigation revealed that the novel hair cells produced, induced by DAPT, were derived from transdifferentiated supporting cells. Additionally, myosin VI‑positive hair cell numbers were increased by Notch signaling inhibition in in vitro experiments with cultured neonatal mouse inner ear precursor cells. This effect was reversed by miR‑183 inhibition. These findings indicate that the Notch signaling pathway served a repressing role during the regeneration of hair cells. Inhibiting this signal improved hair cell regeneration in the gentamicin‑damaged cochlear model. miR‑183 was demonstrated to be involved in hair cell differentiation and regeneration, and was required for the differentiation of the Notch‑inhibited hair cells.