Fucosylation is a post-translational modification that attaches fucose residues to protein- or lipid-bound oligosaccharides. Certain fucosylation pathway genes are aberrantly expressed in several types of cancer, including non-small cell lung cancer (NSCLC), and this aberrant expression is associated with poor prognosis in patients with cancer. However, the molecular mechanism by which these fucosylation pathway genes promote tumor progression has not been well-characterized. The present study analyzed public microarray data obtained from NSCLC samples. Multivariate analysis revealed that altered expression of fucosylation pathway genes, including fucosyltransferase 1 (FUT1), FUT2, FUT3, FUT6, FUT8 and GDP-L-fucose synthase (TSTA3), correlated with poor survival in patients with NSCLC. Inhibition of FUTs by 2F-peracetyl-fucose (2F-PAF) suppressed transforming growth factor β (TGFβ)-mediated Smad3 phosphorylation and nuclear translocation in NSCLC cells. In addition, wound-healing and Transwell migration assays demonstrated that 2F-PAF inhibited TGFβ-induced NSCLC cell migration and invasion. Furthermore, in vivo bioluminescence imaging analysis revealed that 2F-PAF attenuated the metastatic capacity of NSCLC cells. These results may help characterize the oncogenic role of fucosylation in NSCLC biology and highlight its potential for developing cancer therapeutics.