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ObjectivesThere is still controversy about the effect of vitamin D supplementation on osteoarthritis (OA). The purpose of this study was to investigate the effects of vitamin D supplementation with Hyaluronic acid (HA) injection on OA.MethodsWe investigated serum vitamin D levels and oxidative stress (OS) in synovial fluid from patients with OA who underwent total knee arthroplasty (grade IV, n = 24) and HA injection (grade II and III, n = 40). The effects of HA injection with or without oral vitamin D supplementation on synovial fluid OS and knee pain and function were then further investigated. Finally, patients underwent HA injection were divided into two groups according to vitamin D levels (vitamin D < or > 30 ng/ml), and the efficacy of the two groups were compared.ResultsThe results showed that the levels of glutathione peroxidase (GSH-PX) (P < 0.05) in the synovial fluid were lower in patients with stage IV OA than that in patients with stage II-III OA, while the levels of malondialdehyde (MDA) (P < 0.05) and lactate dehydrogenase (LDH) (P < 0.01) were significantly higher. Moreover, we found that age, BMI and vitamin D levels were significantly associated with the levels of oxidants and/or antioxidants in synovial fluid, and that vitamin D was significantly negatively correlated with BMI (R = −0.3527, p = 0.0043). Supplementation of HA injections with vitamin D significantly reduced the OS status in synovial fluid, attenuated knee pain and improved knee function in OA patients with vitamin D insufficiency.ConclusionWe conclude that maintenance of vitamin D sufficiency may be beneficial for the treatment of OA by improving OS in synovial fluid.