Sensorineural hearing loss (SNHL) is one of the most common sensory impairments in humans. However, treatment options mostly rely on medical instruments, with no reliable pharmacological interventions. Reactive oxygen species (ROS) produced by NADPH oxidases (Nox) contribute to the development of different types of acquired SNHL, such as drug-induced HL, age-related HL, and noise-induced HL. Although the essential role of Nox3 in otoconia biosynthesis and its possible involvement in hearing have been reported in rodents, immunohistological methods targeted at detecting Nox3 expression in inner ear cells reveal ambiguous results. Therefore, the mechanism underlying Nox3-dependent SNHL remains unclear and warrants further investigation. We generated Nox3-Cre knock-in mice, in which Nox3 was replaced with Cre recombinase (Cre). Using Nox3-Cre;tdTomato mice, in which tdTomato is expressed under the control of the Nox3 promoter, we identified Nox3-expressing regions and cell types in inner ears. Nox3-expressing cells in cochleae included various types of supporting cells (SCs), outer hair cells (OHCs), inner hair cells, and spiral ganglion neurons. Nox3 expression increased with cisplatin, age, and noise insults in specific cell types in cochleae, and resulted in OHC loss (apoptosis). Moreover, increased Nox3 expression in SCs and OHCs, especially at the basal turn of cochleae, played essential roles in acquired SNHL. Thus, we propose that Nox3 inhibition in cochleae is a promising approach to prevent acquired SNHL.