In hearing loss induced by aminoglycoside antibiotics, the outer hair cells (OHCs) in the basal turn are always more susceptible than OHCs in the apical turn, while the underlying mechanisms remain unknown. In this study, we reported that NAPDH oxidase 2 (NOX2) played an important role in the OHC5 damage preferentially in the basal turn. Normally, NOX2 was evenly expressed in OHC5 among different turns, at a relatively low level. However, after neomycin treatment, NOX2 was dominantly induced in OHC5 in the basal turn. In vivo and in vitro studies demonstrated that inhibition of NOX2 significantly alleviated neomycin-induced OHC5 damages, as seen from both the cleaved caspase-3 and TUNEL staining. Moreover, gp91 ds-tat delivery and DHE staining results showed that NOX2-derived ROS was responsible for neomycin ototoxicity. Taken together, our study shows that regional up-expression of NOX2 and subsequent increase of ROS in OHCs of the basal turn is an important factor contributing to the vulnerability of OHCs there, which should shed light on the prevention of hearing loss induced by aminoglycoside antibiotics. (C) 2018 Elsevier Inc. All rights reserved.