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Rheumatoid arthritis poses a significant threat to the well-being of individuals as an autoimmune disorder. Calycosin is a flavonoid isolated from the root of Astragalus that may be beneficial in treating rheumatoid arthritis. The anti-inflammatory role and mechanism of calycosin were validated in this study through the utilization of collagen-induced arthritis mice models and lipopolysaccharide/interferon gamma stimulated bone marrow-derived macrophages. By applying RNA sequencing technology and the network pharmacological method, it is demonstrated in the findings that the treatment with calycosin leads to a significant alleviation of arthritis symptoms, mitigation of synovial proliferation, impediment of bone and cartilage devastation, and suppression of activated synovial macrophages in collagen-induced arthritis. Mechanistically, activation of JNK and NF-κB pathways is inhibited by calycosin in lipopolysaccharide/interferon gamma stimulated bone marrow-derived macrophages. These results confirm the potential of calycosin, a natural compound, for attenuating rheumatoid arthritis by acting as an anti-inflammatory agent through multiple pathways.