The aim of the present study was to investigate the underlying mechanisms of autophagy in a gentamicin (GM)-induced ototoxic model, and to establish whether the blocking of autophagy significantly increases the survival of inner ear hair cells. Cochleae were carefully dissected from four day‑old C57BL/6J mice and randomly divided into three groups prior to explant culture: Control (culture medium), GM‑treated (culture medium + GM) and GM + 3-methyladenine (3-MA; culture medium + GM + 3‑MA). Transmission electron microscopy, immunofluorescence and western blotting were performed to observe the expression of the autophagy protein microtubule‑associated protein 1A/B‑light chain 3 in explant cultures treated with GM and the autophagy inhibitor 3‑MA. Administration of GM in in vitro mouse cochlear culture induced apoptosis and the formation of autophagic vesicles and autophagosomes in hair cells. Notably, combined treatment with GM and 3‑MA to block autophagy significantly increased the survival of inner ear hair cells. Furthermore, it was indicated that the simultaneous expression and interaction of Atg12 with Bcl‑2 following GM treatment co‑integrated autophagy with apoptosis in the cochlea. The results of the present study demonstrated that autophagy was involved in GM-induced ototoxicity. Additionally, Atg12 may serve a protective role by binding to Bcl‑2. Therefore, Atg12 may be a potential therapeutic target for the treatment of GM-induced cochlear hair loss.