Aminoglycoside antibiotic drugs induce hearing loss in children and adults every year; however, the pathological mechanisms remain unknown. Previous studies have shown that the accumulation of reactive oxygen species (ROS) and inflammation in the inner ear may be responsible for kanamycin (KM)-induced hair cell death and hearing loss. Nucleotide-binding oligomerization domain-, leucine-rich repeat-, and pyrin domain-containing protein 3 (NLRP3) is a specific ROS sensor that initiates inflammasome assembly as well as activates caspase-1 and downstream inflammatory factors. Therefore, this study aimed to determine whether NLRP3 inflammasomes are involved in KM-related hearing loss in mice. Compared with the control (saline) group, increased levels of activated caspase-1, interleukin (IL)-1β, IL-18, N-terminal fragment of gasdermin D (GSDMD-N), and NLRP3 were detected by immunofluorescence, western blot, and enzyme-linked immunosorbent assay (ELISA) in the KM-plus-furosemide (LASIX)-treated group. Moreover, we also found that the NLRP3 inhibitor oridonin (Ori) could significantly rescue KM-related hearing loss by inhibiting NLRP3-inflammasome activation and caspase-1/GSDMD-related hair cell pyroptosis. These findings demonstrate that apoptosis, as well as pyroptosis, may be involved in KM-related hearing loss and that the NLRP3/caspase-1/GSDMD pathway may be a new target for treating aminoglycoside-induced hearing loss.Copyright © 2022. Published by Elsevier Ltd.