Our previous studies showed that cell surface beta 1,4-galactosyltransferase 1 (beta 1,4GT1) negatively regulated cell survival through inhibition and modulation of the epidermal growth factor receptor (EGFR) signaling pathway in human hepatocellular carcinoma (HCC) SMMC-7721 cells. However, the underlying mechanism remains unclear. Here we demonstrated that beta 1,4-galactosyltransferase 1 (beta 1,4GT1) interacted with EGFR in vitro by GST pull-down analysis. Furthermore, we demonstrated that beta 1,4GT1 bound to EGFR in vivo by co-immunoprecipitation and determined the co-localization of beta 1,4GT1 and EGFR on the cell surface via confocal laser scanning microscopy analysis. Finally, using I-125-EGF binding experiments and Western blot analysis, we found that overexpression of beta 1,4GT1 inhibited I-125-EGF binding to EGFR, and consequently reduced the levels of EGFR dimerization and phosphorylation. In contrast, RNAi-mediated knockdown of beta 1,4GT1 increased the levels of EGFR dimerization and phosphorylation. These data suggest that cell surface beta 1,4GT1 interacts with EGFR and inhibits EGFR activation. (C) 2013 Elsevier Inc. All rights reserved.