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Hair cells do not undergo spontaneous regeneration when they are damaged in the mammalian organ of Corti, leading to irreversible hearing loss. Previous studies have shown that 24-diamino-5-phenylthiazole (DAPT), an inhibitor of Notch signaling, plays a major role in inner ear development. However, whether DAPT influences antibiotic-induced hair cell damage remains uncertain. The present study aimed to investigate whether DAPT exerts protective or regenerative effects on neomycin-damaged hair cells. A histological analysis was carried out to assess the number and morphological changes of hair cells in cultured organ of Corti explants. Our results showed that in-vitro treatment with DAPT induced extra hair cells, whereas no newly generated supporting cells were found. We also found that DAPT was effective for preventing hair cell loss when cotreatment with neomycin was performed, suggesting that DAPT exerted protective effects on neomycin ototoxicity. In addition, DAPT treatment for 2–4 days following neomycin damage induced supernumerary hair cells. These findings indicate that inhibition of Notch signaling is a possible strategy for the treatment of hair cell loss caused by aminoglycoside antibiotics.