Osteoarthritis (OA) is a common degenerative joint disease. The pathogenesis of OA is closely related to inflammatory responses and apoptosis of chondrocytes. Hyperoside (Hyp), a natural flavonoid compound, exerts multiple bioactivities in various diseases. Our study aims to investigate the anti-arthritic effects of Hyp and delineate the potential mechanism at the cellular level. Murine chondrocytes were stimulated with interleukin-1β (IL-1β) with or without Hyp treatment. CCK-8 assay was used to evaluate the cytotoxic effect of Hyp. DCFH-DA was used to detect intracellular ROS. Annexin V-FITC/PI method was applied to examine apoptosis of chondrocytes. The anti-arthritic effects of Hyp and related mechanisms were investigated by examining and analyzing relative markers through quantitative PCR, western blot analysis and immunofluorescent staining. C57BL/6 mice were performed the destabilized medial meniscus (DMM) surgery to establish OA model and then injected intraperitoneally with Hyp (20 mg/kg)) for 4 or 8 weeks. Finally, mice were sacrificed and knee joints were collected for histological observation and analysis. Hyp inhibited IL-1β-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Additionally, Hyp attenuated IL-1β-induced destruction of the extracellular matrix (ECM) by downregulating the expression of MMPs and ADAMTS5, and meanwhile upregulating the expression of collagen II, aggrecan, and SOX9. Also, Hyp pretreatment reduced IL-1β-induced overproduction of ROS and apoptosis of chondrocytes. Mechanistically, Hypexerted anti-inflammatory effects by partly suppressing the PI3K/AKT/NF-κB and the MAPK signaling pathways, enhancing the Nrf2/HO-1 to limit the activation of NF-κB. Moreover, Hyp played an anti-apoptotic effect via the Nrf2/ROS/BAX/Bcl-xlaxis. In vivo, cartilage degradation was attenuated with a lower OARSI score in Hyp-treated group compared to the DMM group. Our study demonstrated that anti-arthritic effects of Hyp in vitro and in vivo, indicating Hyp might serve as a potential agent for the treatment of OA. Copyright © 2020. Published by Elsevier GmbH.