Asparagine (N)-linked glycosylation is a protein modification critical for glycoprotein folding, stability, and cellular localization. To identify small molecules that inhibit new targets in this biosynthetic pathway, we initiated a cell-based high-throughput screen and lead-compound-optimization campaign that delivered a cell-permeable inhibitor, NGI-1. NGI-1 targets oligosaccharyltransferase (OST), a hetero-oligomeric enzyme that exists in multiple isoforms and transfers oligosaccharides to recipient proteins. In non-small-cell lung cancer cells, NGI-1 blocks cell-surface localization and signaling of the epidermal growth factor receptor (EGFR) glycoprotein, but selectively arrests proliferation in only those cell lines that are dependent on EGFR (or fibroblast growth factor, FGFR) for survival. In these cell lines, OST inhibition causes cell-cycle arrest accompanied by induction of p21, autofluorescence, and cell morphology changes, all hallmarks of senescence. These results identify OST inhibition as a potential therapeutic approach for treating receptor-tyrosine-kinase-dependent tumors and provides a chemical probe for reversibly regulating N-linked glycosylation in mammalian cells.