The purpose of this study was to use a murine model to determine if melatonin can protect the inner ear from radiation-induced damage. A total of 81 4-week-old Balb/c mice were randomly divided into five groups: control group; 50 mg/kg melatonin group; 5 mg/kg melatonin+radiotherapy group; 50 mg/kg melatonin+radiotherapy group; radiotherapy group. The radiotherapy groups received 16 Gy irradiation and melatonin was administered by intraperitoneal injection 30 min before radiotherapy. On days 3 and 7 after irradiation the function of outer hair cells was determined by auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAEs) testing, pathological changes of inner ear cells were observed by light microscopy, and the expression of prestin mRNA was determined. ABR thresholds were increased and wave I latencies were extended after radiotherapy; however, the increases were lower in the groups that received melatonin (P < 0.05). DPOAEs showed radiotherapy-induced hearing loss at 8-12 kHz, and hearing loss was greater on day 7 than day 3. However, hearing loss was less in the melatonin groups (P < 0.05). Histopathological examination showed irradiation resulted in breaks and distortion of the cochlear basement membrane, disruption of the stria vascularis, and swelling of outer hair cells. Melatonin reduced these changes. Radiotherapy upregulated prestin mRNA expression. Radiotherapy-induced upregulation of prestin was decreased in the melatonin groups (P < 0.05), and the decrease was greater in the 50 mg/kg melatonin group (P < 0.05). Melatonin protects against radiation-induced cochlear damage by reducing damage to outer hair cells.