Clinically used gentamicin, a widely used aminoglycoside antibiotic, is a mixture of 5 major subtypes with potentially differential contributions to the drug’s known ototoxicity. The gentamicin subtype C2b has been previously associated with reduced ototoxicity in animal models in vitro, but its effects in humans remain uncertain due to the lack of appropriate in vitro models of the human inner ear. To address this gap, we generated otic progenitor cells (OPCs) from human induced pluripotent stem cells and used transcriptomic and immunocytochemical analyses to confirm their otic lineage identity and validate their relevance as a model for ototoxicity studies. We then compared the effects of gentamicin C2b and clinical gentamicin on cell viability and cytotoxicity in human OPCs. In parallel, we examined auditory function in mice following exposure to each formulation using auditory brainstem response and distortion product otoacoustic emissions. Gentamicin C2b exposure resulted in substantially lower cytotoxicity—via reduced mitochondria-dependent apoptosis—and higher cell viability in human OPCs, as well as dramatically attenuated hearing loss across all frequencies in mice. Together, these findings indicate that gentamicin C2b has reduced ototoxicity compared to clinically used preparations, supporting the need for further investigations in clinical settings.