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To observe the effect of melatonin intervention on rat knee osteoarthritis (KOA) model and explore its mechanism.A total of 81 Sprague-Dawley (SD) rats were employed. Haematoxylin and eosin (HE) staining and safranin o-solid green staining were used to observe the changes of pathology in KOA, and inflammation factors in serum were detected by enzyme-linked immunosorbent assay (ELISA), type II collagen (Col-II) was detected by immunohistochemistry, chondrocyte apoptosis was detected by TdT-mediated dUTP nick-end labeling (TUNEL). The expression of matrix metalloproteinases (MMPs) and JAK2/STAT3 signaling were detected by western blot.Melatonin treatment ameliorated the histomorphology of knee joint in rats compared to the model group. The contents of TNF-α, IL-6, and IL-1β in serum were decreased after melatonin treatment. In addition, compared to the model group, the positive expression of Col-II increased, the chondrocyte apoptosis decreased after melatonin treatment. Interestingly, the expression levels of MMP3, MMP9, MMP13, p-JAK2 and p-STAT3 decreased (p 0.05). Importantly, melatonin combined with AG490 is significantly ameliorates histomorphology of knee joint, reduced cartilage loss compared with melatonin treatment alone.Melatonin treatment can effectively diminish the cartilage injury. Its mechanism may be related to protect the articular cartilage by reducing the release of inflammatory factors, inhibit the expression of MMPs and JAK2/STAT3 signaling.