Background Oxymatrine (OMT) is one of the authentic Chinese herbal medicines which has rich and complex active ingredients. however,the relevant potential targets of oxymatrine on rheumatoid arthritis and the mechanism remains unreported. The aim of this study was to determine the regulation of oxymatrine on rheumatoid arthritis using a collagen‐induced arthritis (CIA) mice models and blood samples from RA patients. Results Our results indicated that Tfr cells in RA patients express low levels of Blimp‐1 and CTLA‐4. Oxymatrine treatment of CIA mice alleviated joint swelling, reduced the arthritis score, and improved joint damage. While flow cytometry results showed that oxymatrine treatment reduced Tfh cells and B cells, and increased Tfr cells in the spleen of CIA mice. In addition, oxymatrine treatment significantly down‐regulated the expression of TLR9（Toll‐like Receptors 9), IL‐21, MyD88, STAT3, p‐STAT3, and CXCR5 in the synovial tissues of CIA mice, and up‐regulated the expression of Foxp3, Blimp‐1, and CTLA‐4. Conclusion Oxymatrine can alleviate rheumatoid arthritis by regulating the TLR9‐MyD88‐STAT3 signaling pathway to maintain immune balance between Tfr‐Tfh cells. © 2023 Society of Chemical Industry.