Lewis Y (LeY) antigen is an oligosaccharide that is highly expressed at the cell surface in various human cancers. Increased LeY expression activates epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) and promotes cell proliferation in EGFR-overexpressing cells. However, the effect of downregulation of LeY expression on cell proliferation in HER2-overexpressing cells remains unknown. FUT1 encodes α1,2-fucosyltransferase, a key enzyme for LeY synthesis. We knocked down FUT1 by short interfering RNA (siRNA) in four HER2-overexpressing human cancer cell lines, including NCI-N87, MKN7, SKBr3 and BT474. We investigated whether downregulation of LeY and alteration in the glycosylation status of these cells affect cell proliferation and HER2 activation. Knocking down FUT1 expression markedly inhibited proliferation of NCI-N87, which highly expressed EGFR and was sensitive to EGFR deprivation. Furthermore, FUT1 siRNA downregulated the total amount of HER2 protein, phosphorylation of HER2 and EGFR, and phosphorylation of extracellular signal-regulated kinase (ERK) in this cell line. Moreover, the marked downregulation of phosphorylation of HER2 and ERK was observed following short-time EGF-stimulation. These effects were not observed in the other three cell lines. Our results suggest that knockdown of FUT1 downregulates HER2 signaling via EGFR downregulation. FUT1 may serve as a new molecular target for HER2-overexpressing human cancers with activated EGFR signaling.