Rheumatoid arthritis (RA) is a chronic autoimmune disease with high morbidity and mortality. Fibroblast-like synoviocytes (FLS) in the synovial tissues play critical roles in joint destruction. Recent studies implicate the sumoylation in the regulation of the inflammation and arthritis. Thus, we explored whether SUMO-conjugating enzyme UBC9 is involved in the progression of RA using a mouse collagen-induced arthritis (CIA) model. The effects of UBC9 siRNA on cell invasion and migration in human RA-FLS were also assessed in vitro. Treatment with siRNA against UBC9 for 3 weeks reduced the arthritis score and joint destruction. The expression of SUMO-1 and UBC9 protein in CIA joints was inhibited by UBC9 knockdown. Serum levels of anti-collagen (CII) antibodies, vascular endothelial growth factor A (VEGF-A), matrix metalloproteinases (MMP)-3, and MMP-9 were also decreased in CIA mice. In vitro, UBC9 silencing inhibited the secretion of VEGF-A, MMP-3, and MMP-9 from TNF-α-stimulated human RA-FLS. TNF-α-induced RA-FLS proliferation and migration were significantly attenuated by UBC9 knockdown. These findings indicate that SUMO-conjugating enzyme UBC9 promotes proliferation and migration of fibroblast-like synoviocytes in rheumatoid arthritis. Inhibition of UBC9 activity may be a viable therapeutic target in amelioration of disease progression in RA by attenuating FLS proliferation, migration, and invasion.