This study aims to explore the mechanism of isoquercitrin (IQC) in treating experimental rheumatoid arthritis (RA) using transcriptomics analysis combined with cell experiments. Firstly, the potential targets of IQC and RA were screened using network pharmacology. Then, the RAW 264.7 cell inflammation model induced by lipopolysaccharide (LPS) was used for effective concentration screening of IQC and transcriptomic analysis of potential targets for IQC treatment of RA. Subsequently, core targets were revealed via intersection analysis of targets identified by both methods, and molecular docking simulations using AutoDock were performed to verify the binding affinity between IQC and core target. Finally, LPS was used to induce macrophages and fibroblast like synoviocytes (FLS) to construct inflammatory models for experimental validation. The results of network pharmacology showed that there were 230 and 2 980 targets for IQC and RA, respectively. Transcriptome sequencing analysis identified 101 significant differentially expressed genes. Through intersection analysis, tyrosine protein kinase receptor TYRO3 (TYRO3) was ultimately determined as the key target, and the binding energy between IQC and TYRO3 was -7.1 kcal/mol. The cell experiment results showed that the effective concentration of IQC was 0.25-25 μmol/L, and IQC could inhibit the accumulation of reactive oxygen species (ROS) in RAW 264.7 cells. RT-qPCR and western blot results showed that IQC could significantly downregulate the gene expression levels of pro-inflammatory cytokine such as interleukin-1β (IL-1β), IL-6, inducible nitric oxide synthase (iNOS) and significantly upregulate the gene expression levels of anti-inflammatory cytokine such as arginase-1 (ARG-1) and IL-10 by inhibiting the mRNA level of TYRO3 in macrophages, significantly downregulate the protein expression levels of TYRO3, IL-1β, tumor necrosis factor-α (TNF-α), and significantly upregulate the protein expression level of IL-10, achieving the effect of inhibiting the inflammatory response of RAW 264.7 cells. In addition, IQC could significantly inhibit the migration and invasion ability of FLS cells and significantly downregulate the mRNA expression levels of matrix metalloproteinase-1 (MMP-1), MMP-3, MMP-9, IL-1β, IL-6, TNF-α, as well as the protein expression levels of TYRO3 and MMP-9 in FLS cells, achieving the effect of inhibiting the inflammatory response and invasion ability of synovial fibroblasts. In summary, IQC may alleviate macrophage inflammation by regulating TYRO3 receptor signaling, block key pathways for abnormal activation of fibroblasts, and exert multi-target anti RA effects.