Disease-modifying antirheumatic drugs (DMARDs) are the first line medications to treat rheumatoid arthritis (RA), a chronic and systemic autoimmune disease affecting multiple joints. Sinomenine (SIN) is thought a natural DMARD (nDMARD) and effectively utilized to treat RA in clinic for several decades in China. Here we reported that it is not methotrexate (MTX), a representative drug of DMARDs, but SIN protected joints from destruction to alleviate the symptoms of the mice with arthritis, indicating that the underlying mechanism of SIN is different from MTX to treat arthritis. Due to the dominate role of synovium fibroblasts in the joint destruction of arthritis, we applied synovium fibroblasts derived from RA patients (RASFs) to investigate the anti-arthritic effect and explore the underlying mechanism of SIN. We found that SIN significantly inhibited the secretion of IL-6 and IL-33 and ROS production in RASFs to mediate protective effect on bone destruction to mediate anti-arthritis effect. Underlying mechanistic study showed that SIN induced phosphorylation of p62 at Ser349 and Thr269/Ser272 to activate Keap1-Nrf2 signaling in RASFs. In line with the results, we then observed that the anti-arthritic effect of SIN was significantly attenuated in Nrf2 deficient (Nrf2−/−) mice. Notably, we found that p62 expression and phosphorylation at Thr269/Ser272 remarkably reduced, while p62 phosphorylation at Ser351 was up-regulated in Nrf2 deficient mice compared to its wild littermates, indicating that Nrf2 probably negative regulates p62 phosphorylation at Ser351. Collectively, our findings demonstrate that SIN phosphorylated p62 at Ser351 (corresponding to human Ser349) to degrade Keap1 expression and accumulate Nrf2 expression, increased p62 expression and phosphorylation at Thr269/Ser272 to activate p62-Keap1-Nrf2 axis, and finally exerted anti-arthritic effect. The current study not only clarified the anti-arthritic characteristics of SIN but also provided the clue to elucidate the correlation of p62 phosphorylation sites and Nrf2 signaling activation. © 2020