Abstract Inflammatory macrophages (MΦ) play an important role in rheumatoid arthritis (RA). Fucosylation is a regulator of inflammation and we have observed a highly positive correlation between Fucosyltransferases (Fut)1 or Fut3 and Tnfα (p=0.0001) in RA synovial tissues. In sorted cells from human RA synovial fluid, Fut1 and Fut3 was highly expressed in M1 inflammatory MΦ (CD68+CD80+), but not in M2 MΦ (CD68+CD80-), synovial fibroblasts, Th1, Th17, and other T cells (p<0.01, n=5). Fucosylation inhibitor, 2-Deoxy-D-galactose (2-D-gal), precluded the differentiation of M1 MΦ derived by GM-CSF from mouse bone marrow or human PBMC monocytes, strongly indicating the indispensable role of fucosylation in M1 MΦ differentiation. Additionally, phalloidin staining showed 2-D-gal disrupted MΦ actin, suggesting a potential effect of fucosylation in antigen processing and presentation. Indeed, 2-D-gal treatment of fully differentiated M1 MΦ for 2 days dramatically reduced their uptake, processing and presentation of GFP-Eα (from I-Edα) and FITC-CII antigens in a dose dependent manner (p<0.01). In vivo, 2-D-gal dramatically blocked the bovine CII-induced arthritis in DBA/1J mice (scores 9.5±1.7 vs 0.5± 0.3, p<0.01) with reduced inflammatory MΦ in draining LN (1.3±0.3% vs 0.5±0.1%, p<0.05), decreased TNF-α (130 vs 39 pg/ml, p<0.05), and anti-CII in the serum. Our study indicated that M1 MΦ differentiation and function is orchestrated by fucosylation, which is a novel therapeutic target in RA.