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Osteoarthritis (OA) is characterized by progressive cartilage destruction and synovitis; however, there are no approved disease-modifying OA drugs. Krill oil (KO) has been reported to possess anti-inflammatory properties and alleviate joint pain in knee OA, indicating its potential to target the inflammatory mechanism of OA. Therefore, the anti-OA effects of KO were investigated in primary chondrocytes and a surgical rat model of knee OA. The oral administration of KO at 200 and 100 mg/kg for 8 weeks improved joint swelling and mobility in the animal model and led to increased bone mineral density and compressive strength in the cartilage. The oral KO doses upregulated chondrogenic genes (<i>type 2 collagen</i>, <i>aggrecan</i>, and <i>Sox9</i>), with inhibition of inflammation markers (5-lipoxygenase and prostaglandin E₂) and extracellular matrix (ECM)-degrading enzymes (MMP-2 and MMP-9) in the cartilage and synovium. Consistently, KO treatments increased the viability of chondrocytes exposed to interleukin 1α, accompanied by the upregulation of the chondrogenic genes and the inhibition of the ECM-degrading enzymes. Furthermore, KO demonstrated inhibitory effects on lipopolysaccharide-induced chondrocyte inflammation. Histopathological and immunohistochemical analyses revealed that KO improved joint destruction and synovial inflammation, probably due to the anti-inflammatory, anti-apoptotic, and chondrogenic effects. These findings suggest the therapeutic potential of KO for knee OA.