〔Abstract〕 ObjectiveTo investigate the protective effect of genistein (Gen) on etoposide-induced chondrocyte senescence and its underlying mechanism. Methods The C28/I2 cell line was treated with different concentrations of Gen and etoposide, and the cell viability was detected by the CCK-8 assay. The senescence model of C28/I2 chondrocytes was induced by etoposide, with Gen intervention. Senescence-associated β-galactosidase (SA-β-gal) staining was performed to detect the senescence-positive rate and staining characteristics of chondrocytes. The expressions of peroxiredoxin 6 (Prdx6), cyclin-dependent kinase inhibitor 1A (p21), and cyclin-dependent kinase inhibitor 2A (p16) were detected by Western blot, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and immunofluorescence staining. The activity of glutathione peroxidase (GPx) was determined using a glutathione peroxidase assay kit. The direct binding between Gen and Prdx6 was verified by molecular docking. Finally, Prdx6-siRNA silencing experiment was performed to clarify the functional necessity of Prdx6. ResultsCompared with the etoposide group, the C28/I2 chondrocyte viability significantly increased ( P<0.01), the expression of senescence-associated proteins p21 and p16 decreased ( P<0.01, P<0.05), the expression of senescence-associated genes p21 and p16 reduced (both P<0.01), the fluorescence intensity of senescence-associated proteins p21 and p16 was diminished ( P<0.05, P<0.01), and the proportion of SA-β-gal-positive cells decreased ( P<0.01) in the Gen+etoposide group. Compared with the Control group, the expression of Prdx6 was downregulated in the etoposide group ( P<0.05). Compared with the etoposide group, the expression of Prdx6 was upregulated in the Gen+etoposide group ( P<0.01). Compared with the Control group, the GPx activity significantly decreased in the si-Prdx6 group ( P<0.01). Furthermore, compared with the si-Prdx6 group, the GPx activity increased in the si-Prdx6+Gen group ( P<0.05). Molecular docking results revealed that Gen formed hydrogen bond interactions with the active site of Prdx6. After Prdx6 knockdown, the expression of senescence-associated genes p21 and p16 and the fluorescence intensity of senescence-associated proteins p21 and p16 both increased in the Gen+etoposide+si-Prdx6 group (both P<0.01). Conclusion Gen can inhibit etoposide-induced senescence of C28/I2 chondrocytes by upregulating the expression of Prdx6. This study provides potential drug targets and experimental basis for the prevention and treatment of chondrocyte senescence-related diseases.