Age-related hearing loss (AHL) is the most common sensory disorder of aged population. Currently, one of the most important sources of experimental medicine for AHL is medicinal plants. This study performed the first investigation of the effect of thymoquinone (TQ), a potent antioxidant, on AHL. Here, we used inbred C57BL/6J mice (B6 mice) as a successful experimental model of the early onset of AHL. The behavioral assessment of hearing revealed that the injection of a high dose of TQ (40 mg/kg; TQ₄₀) significantly improved the auditory sensitivity of B6 mice at all tested frequencies (8, 16 and 22 kHz). Histological sections of cochlea from B6 mice injected with a low dose (20 mg/kg; TQ₂₀) and high dose showed relatively less degenerative signs in the modiolus, hair cells and spiral ligaments, the main constituents of the cochlea. In addition, TQ₄₀ completely restored the normal pattern of hair cells in B6 mice, as shown in scanning electron micrographs. Our data indicated that TQ₂₀ and TQ₄₀ reduced levels of Bak1-mediated apoptosis in the cochlea of B6 mice. Interestingly, the level of Sirt1, a positive regulator of autophagy, was significantly increased in B6 mice administered TQ₄₀. In conclusion, TQ relieves the symptoms of AHL by downregulating Bak1 and activating Sirt1 in the cochlea of B6 mice.