OBJECTIVE Glycine tabacina (Labill.) Benth (family: Leguminosae), named Yan Dou in Chinese, is mainly distributed in the coastal areas of subtropical and tropical areas such as southern China, Taiwan, eastern Australia and some South Pacific Islands. Its root has been used to treat rheumatoid arthritis (RA) and joint infection for many years in folk medicine. However, the chemical constituents and pharmacology of this species are largely unknown. This study aimed to reveal the anti-arthritic constituents and to investigate the anti-arthritic effects and underlying mechanisms of G. tabacina by the integration of network pharmacology and experimental pharmacology. RESULTS We found that oral administration of ethanol extract of G. tabacina (GTE, 1.11-4.44 g dry weight of herb powder per kg body weight) significantly alleviated paws swelling, decreased arthritis index, inhibited bone erosion and cartilage destruction, decreased pro-inflammatory cytokines production (IL-1β, IL-6 and TNF-α) and oxidative stress in collagen-induced arthritis (CIA) rat model. Current phytochemical research on this herb led to the isolation of eight new coumestans, named glytabastan A-H (1-8), together with twenty-three known compounds 9-31. Their structures were elucidated using various spectroscopic methods. The anti-arthritic activities of all isolates were evaluated, and the results showed that the anti-arthritic activity of G. tabacina might be attributed to coumestans and isoflavonoids in this species. Particularly, coumestans 1-6 and 8-10 could inhibit arthritic inflammation in vitro, while coumestans 1, 2, 9 and 10 significantly blocked the osteoclastogenesis induced by receptor activator of nuclear factor (NF) κB ligand (RANKL). Network pharmacology analysis revealed that 65 candidate targets (AKT1, STAT3, IL6, INS, VEGFA, JUN, MAPK1, EGFR, et al.) of GTE were related to its therapeutic effect on RA, and the GO and KEGG pathway enrichment analysis revealed that these candidate targets were significantly involved in forty-one biological processes (inflammatory response, cytokine-mediated signaling pathway, reactive oxygen species metabolic process, et al.) and thirty-one signal pathways (PI3K/AKT pathway, MAPK pathway, HIF-1 pathway, et al.). These results revealed that the regulation of GTE on RA could be via acting on multi-targets, multi-pathways and multiple biological processes associated with RA. Further, experimental pharmacology proved that coumestan 9 (or named Dolichosin A, DoA) could exhibit strong anti-arthritic activity through suppressing PI3K/AKT and MAPK pathways activation in activated synovial cells and osteoclasts. Coumestan 2 (i.e. glytabastan 2, G2) significantly inhibited IL-1β-induced expression of pro-inflammatory mediators and matrix metalloproteinases (MMPs) in SW982 human synovial cells, suppressed RANKL-induced differentiation of bone marrow-derived macrophages (BMMs) towards to mature osteoclasts and the bone resorption function of osteoclasts, and ameliorated the arthritic symptoms in CIA mice at dose of 12.5 and 25 mg/kg. Phosphoproteomic analysis of G2-treated SW982 cells indicated that the anti-arthritic effect of G2 was closely associated with the regulation of various pathways including spliceosome, pathways in cancer, PI3K-Akt signaling pathway, RNA transport, regulation of actin cytoskeleton, MAPK signaling pathway, apoptosis and Rap1 signaling pathway. CONCLUSION These results suggested that the potential applications of G. tabacina and its active natural products especially new coumestans in RA prevention and treatment as functional food or therapeutic agents. This study additionally provided important chemical information for the development of novel agent(s) for the treatment of other inflammatory diseases from G. tabacina.