BACKGROUND: Cisplatin (CP) has been used as an effective chemotherapy drug for different types of cancers. Despite its therapeutic benefits, the clinical utility of CP is often hindered by adverse effects, notably acute kidney injury (AKI), which restricts its widespread application. Dihydromyricetin (DHM) is a flavonoid acquired from Ampelopsis grossedentata, exhibiting a range of pharmacological activities. The major objective of this research was to examine the possible molecular mechanism involved in CP-induced AKI and the protective function of DHM. METHODS: In this study, the protective function of DHM against CP-induced AKI was assessed in both mice and HK-2 cells. Kidney dysfunction parameters and renal morphology were evaluated to ascertain the extent of protection. Additionally, proteomics techniques were employed to investigate the protective effect of DHM and elucidate the underlying molecular mechanisms involved in mitigating CP-induced AKI. In addition, protein levels of epidermal growth factor receptor (EGFR), p-EGFR, heat shock protein 27 (HSP27), p-HSP27, STAT3, and p-STAT3 in renal tissues were investigated. Furthermore, an EGFR-blocking agent (gefitinib) or si-RNA of HSP27 was used to study the effects of inhibiting EGFR or HSP27 on CP-induced renal injury. RESULTS: DHM decreased blood urea nitrogen (BUN) and creatinine in serum, alleviated renal morphological injury and downregulated the expression of CP-induced kidney injury molecule-1 and neutrophil gelatinase-related lipocalin. Proteomic data revealed HSP27 as a potential therapeutic target for AKI. DHM treatment resulted in the downregulation of EGFR, HSP27, and STAT3 phosphorylation, ultimately mitigating CP-induced AKI. In addition, the inhibition of EGFR or HSP27 reduced mitochondria-mediated apoptosis and CP-induced cell damage in HK-2 cells. CONCLUSIONS: DHM effectively inhibited CP-induced oxidative stress, inflammation, and mitochondria-mediated apoptosis through the EGFR/HSP27/STAT3 pathway.