〔Abstract〕 Objective To establish anin vitrorenal injury model of amikacin(AKN) and investigate the protective effect and mechanism of vaccarin(VA) in the AKN-inducedin vitrorenal injury model. Methods Human renal tubular epithelial cells(HK-2) were culturedin vitroand incubated with different drugs of AKN or/and VA to determine the optimal drug concentration based on cell viability tested by MTT. The changes in intracellular oxidative stress were assessed using the dihydroethidium(DHE) probe and malondialdehyde(MDA)/glutathione(GSH) assay kits at different time points. Total RNA was extracted, and RT-qPCR was performed to detect the changes in the gene expression of kidney injury molecule-1(KIM-1) and neutropil gelatinase-associated lipocalin(NGAL). Western blot analysis was performed to detect the levels of ferroptosis-related markers solute carrier family 7 member 11(SLC7A11) and glutathione peroxidase 4(GPX4) in HK-2 cell lysis. Results High concentrations of AKN significantly decreased the viability of HK-2 cellsin vitro, with a half maximal inhibitory concentration(IC50) of(5.74±0.47) mmol/L. VA at concentrations of 25-100 μmol/L increased the viability of AKN-stimulated HK-2 cells(P<0.05). After treatment with AKN(4 mmol/L), the mRNA expression levels of KIM-1 and NGAL were significantly higher than those of the negative control(NC) group(P<0.001). VA(50 μmol/L) significantly reduced the mRNA expression levels of KIM-1(P<0.01) and NGAL(P<0.05). The intensity of DHE staining increased after 3 hours of AKN treatment, but the difference was not statistically significant. However, the intensity of DHE staining was significantly higher in the 6-24 hours group compared to the 0-hour group(P<0.01). Furthermore, MDA levels significantly increased, while GSH levels significantly decreased after 6-24 hours of AKN treatment, with statistically significant differences(P<0.05). After 6-24 hours of AKN stimulation, the ferroptosis-related proteins SLC7A11 and GPX4 both significantly decreased(P<0.001). Co-incubation with VA for 24 hours effectively reversed the changes in DHE staining, MDA and GSH levels, as well as the changes of SLC7A11 and GPX4 protein levels(P<0.001). Conclusion In this study, anin vitrorenal injury model was established by stimulating HK-2 cells with high concentrations of AKN, and it was found that VA might alleviate the damage to renal tubular cells caused by AKNviainhibiting oxidative stress related ferroptosis.