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Osteoarthritis (OA), a chronic disease featured by severe pain and persistent inflammation, significantly affects human health and lowers the quality of life. Nuciferine (Nuf) is major alkaloids abundant in lotus leaf and has various biological activities. In current study, we aimed to explored the protective effect of Nuf against OA and its underlying mechanism in vitro and in vivo. It was found that Nuf alleviated the degradation of extracellular matrix (ECM) under the stimulation of IL-1β and inhibited the production of inflammation cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in vitro. Meanwhile, it was demonstrated that Nuf suppressed the activation of PI3K/Akt/NF-κB signaling pathway induced by IL-1β. Besides, there was favorable binding ability between Nuf and PI3K. Nuf significantly alleviated the development of OA in vivo in the rat OA model established by anterior cruciate ligament transaction (ACLT). Together, our results indicated that Nuf attenuates the progression of OA by targeting PI3K/Akt/NF-κB signaling pathway, which makes Nuf as a promising candidate for the treatment of OA.