Cisplatin-induced hearing loss (CIHL) affects half a million cancer patients annually, but treatment strategies are limited. We have previously shown that honokiol, an antioxidant and otoprotective compound, upregulates mitochondrial sirtuin 3 (SIRT3) and protects against CIHL. In the present study, we investigated the molecular requirements of this protection using a Sirt3 knockout ( Sirt3 ^−/− ) mouse model. Our results showed that honokiol pre-treatment ameliorated auditory threshold shifts in both wild-type and Sirt3 ^−/− mice. Quantitative histological analysis showed that honokiol pre-treatment protected against cisplatin-induced synaptopathy. To explore the underlying molecular mechanisms, we performed RNA–seq on wild-type outer hair cells (OHCs). Cisplatin treatment resulted in a global transcriptomic downregulation, which was biased towards longer genes. Differential expression analysis showed that cisplatin treatment resulted in the downregulation of mitochondrial redox genes, such as Idh2 and Aldh2 , a pattern that was reversed by honokiol pre-treatment. Computational transcription factor binding site analysis predicted that honokiol rescues chromatin-modifying pathways, while suppressing the ternary complex factor subfamily. Lastly, honokiol-only treatment proactively reinforced antioxidant defenses via Sod2 upregulation. Together, these results provide a comprehensive transcriptomic map of OHCs in the cisplatin background and highlight the clinical potential of honokiol to protect against CIHL.