β1,4-Galactosyltransferase-I (β1,4-GalT-I), which transfers galactose to the terminal N-acetylglucosamine of N- and O-linked glycans in a β1,4-linkage, is considered to be the major galactosyltransferase among the seven members of the subfamily responsible for β4 galactosylation. We previously reported, for the first time, that β1,4-GalT-I may play an important role in the inflammatory processes in synovial tissue of patients with rheumatoid arthritis (RA). In this study, we analyzed whether β1,4-GalT-I expression correlates with the expression of tumor necrosis factor-α (TNF-α) in RA. We show firstly the overexpression and co-localization of β1,4-GalT-I and TNF-α in synovial tissue of RA patients. Then, lipopolysaccharide (LPS) induces β1,4-GalT-I mRNA up-regulation in fibroblast-like synoviocytes (FLSs) through endogenous TNF-α overexpression. In addition, we observed that not only endogenous TNF-α but also exogenous TNF-α induced β1,4-GalT-I mRNA production in FLSs, and TNF-α-knockdown reverses the up-regulation of β1,4-GalT-I in FLSs induced by LPS or TNF-α. These results suggest that TNF-α contributes to the up-regulation of β1,4-GalT-I mRNA in human FLSs.