Objective To investigate the protective effect of overexpression of Optic Atrophy Protein 1 (OPA1) on interleukin-1β (IL-1β)-induced damage in human chondrocytes through Parkin-mediated mitophagy. Methods C28/I2 cells were transfected with Ad-OPA1, Ad-null, siRNA, and si-OPA1, respectively, and then treated with IL-1β(5 ng/ml) for 24 hours. Western blot was used to analyze the expression of OPA1 in chondrocytes, MTT assay was used to detect cell viability, and flow cytometry was used to measure the apoptosis rate. Western blot was also used to measure the expression of Caspase-3, B-cell lymphoma-2 (Bcl-2), and Bcl-2 associated X protein (BAX), as well as the expression of Collagen Ⅱ, Aggrecan, and Matrix metalloproteinase 13 (MMP-13). Additionally, the levels of reactive oxygen species (ROS), glutathione peroxidase (GPX), and superoxide dismutase (SOD) activity were detected. JC-1 probe was used to measure mitochondrial membrane potential (ΔΨM), and mitochondrial ATP kit was used to measure adenosine triphosphate (ATP) content. RT-qPCR was used to detect the mRNA expression of mitochondrial dynamics markers PGC-1α, MFN1, and Drp1. Western blot was used to measure the expression levels of P62, LC3B, and mitophagy markers Beclin-1, LAMP1, PINK1, Parkin, and USP30 in chondrocytes. C28/I2 cells were transfected with Ad-OPA1 and Parkin-siRNA, and Western blot was used to detect the protein expression of Parkin, P62, LAMP1, Beclin-1, PINK1, USP30, cleaved-caspase3, Aggrecan, Collagen Ⅱ, and MMP-13. The expression levels of target proteins, ROS levels, SOD activity, membrane potential depolarization, cell viability, and apoptosis rate were also measured. Results ter IL-1β treatment of adenovirus-transfected cells, Western blot results showed a significant decrease in OPA1 protein expression, while Ad-OPA1 increased OPA1 expression. MTT results indicated that OPA1 overexpression enhanced cell viability inhibited by IL-1β. Flow cytometry results showed that OPA1 overexpression inhibited chondrocyte apoptosis. Further Western blot results showed that OPA1 significantly reduced the protein levels of Caspase3 and BAX and upregulated Bcl-2 expression in chondrocytes. OPA1 also upregulated Collagen Ⅱ and Aggrecan expression while reducing MMP-13 expression. OPA1 overexpression decreased ROS levels, increased GPX and SOD activity, and enhanced ΔΨM and ATP content (which were reduced by IL-1β). RT-qPCR results showed that OPA1 increased the mRNA expression of PGC-1α and MFN1 while downregulating Drp1 mRNA expression. Western blot results showed that OPA1 downregulated P62 and USP30 expression and upregulated LC3B and mitophagy markers Beclin-1, LAMP1, PINK1, and Parkin. After transfection of C28/I2 cells with Parkin-siRNA, OPA1-induced Parkin expression was significantly downregulated. Parkin-siRNA increased ROS, P62, USP30, Caspase3, and MMP-13 expression, while reducing GPX and SOD activity, ΔΨM, ATP content, Collagen Ⅱ, Aggrecan, LC3B, and mitophagy markers Beclin-1, LAMP1, PINK1, and Parkin protein expression. Additionally, Parkin-siRNA significantly reduced cell viability and increased chondrocyte apoptosis rate. Conclusion OPA1 protects chondrocytes from damage and extracellular matrix (ECM) degradation through Parkin-mediated mitophagy, thereby alleviating IL-1β-induced mitochondrial dysfunction and oxidative stress, and providing a protective effect on OA articular cartilage