In this study, we probed into the related mechanism underlying the role of Tanshinone IIA (TIIA) in RA fibroblast-like synoviocytes (RA-FLSs). We constructed a mouse model of RA using the collagen-induced arthritis (CIA) method. Gain- or loss-of-function approaches were used to manipulate matrix metalloproteinase9 (MMP9), receptor for advanced glycation end product (RAGE), and toll-like receptor 9 (TLR9) in both CIA mice and RA-FLSs following treatment with TIIA to study the in vivo and in vitro effect of TIIA through analysis of cell viability, and measurement of autophagy and inflammatory proteins as well as severity of RA. In vitro and in vivo animal experiments results showed that TIIA could inhibit the proliferation of RA-FLSs and affect autophagy, thereby improving the symptoms of RA in mice. Mechanically, TIIA could inhibit the expression of MMP9 in RA-FLSs, thereby inhibiting the shedding of RAGE and thus inhibiting the activation of TLR9. Finally, animal experiments confirmed that TIIA affected autophagy by regulating the MMP9/RAGE/TLR9 axis, and finally improve the symptoms of RA in mice. Conclusively, TIIA may inhibit expression of MMP9 to suppress the combination of RAGE and TLR9, thereby inhibiting RA-FLS proliferation and affecting autophagy, eventually improve the RA., (© 2023 The Authors. Phytotherapy Research published by John Wiley & Sons Ltd.)