Background: Rheumatoid arthritis (RA) is a chronic inflammatory disease with a high rate of disability accompanied by various complications. The pathogenesis of RA is complex with multiple targets and links. This study aims to investigate pharmacological effects and mechanism of Rhodojaponin III in RA. Methods: The bovine type II collagen-induced arthritis (CIA) rat model and tumor necrosis factor-alpha (TNF-\alpha) induced human umbilical vein endothelial cells (HUVECs) model were constructed. Different concentrations of Rhodojaponin III were utilized for intervention. The progression of CIA was assessed by the arthritis index (AI). Pathological changes in knee joints and synovium were observed. The expressions of angiogenesis-related cytokines were detected. The proliferation, migration, invasion, and angiogenesis of HUVECs were detected. The levels of pro-inflammatory cytokines were determined. The expressions of nuclear factor kappa B-inducing kinase (NIK) pathway-related proteins were analyzed. The binding of Rhodojaponin III to NIK was simulated by molecular docking. Results: Rhodojaponin III suppressed cartilage damage and bone erosion in the knee joints. Rhodojaponin III inhibited expressions of platelet endothelial cell adhesion molecule-1 (CD31) and vascular endothelial cell growth factor (VEGF) to decrease vascular density. Rhodojaponin III suppressed the proliferation, migration, invasion, and angiogenesis of HUVECs, and decreased the levels of interleukin (IL)-6, IL-1\beta, and TNF-\alpha. Molecular docking showed that Rhodojaponin III could spontaneously bind to NIK. Rhodojaponin III decreased the expression of NIK, p52, and C-X-C motif chemokine ligand 12 (CXCL12) and the phosphorylation level of I\kappaB kinase-alpha (IKK\alpha) in the synovium of CIA rats and TNF-\alpha-induced HUVECs. NIK overexpression reversed the inhibitory effect of Rhodojaponin III on activation of the NIK/NF-\kappaB pathway, migration, invasion, and angiogenesis of HUVECs, and the secretion of pro-inflammatory cytokines. Conclusions: Rhodojaponin III affected the angiogenesis and inflammation of CIA rats and TNF-\alpha-induced HUVECs by regulating the NIK/IKK\alpha/CXCL12 pathway. These findings suggest that Rhodojaponin III has potential as a therapeutic agent for RA. Further studies are needed to explore its precise mechanism of action and evaluate its clinical efficacy.