The key post-translational modification of protein N-glycosylation helps proteins fold, regulates the functions of mature glycoproteins, and is important in essentially all biological processes involving cell-cell or protein-cell interactions. As a key cellular process affecting most secreted and membrane proteins, perturbations to the normal regulation of glycosylation can affect diverse proteins. To study the physiological regulation of protein glycosylation we have used Data-Independent Acquisition Liquid Chromatography Mass Spectrometry (DIA LC-MS/MS) in combination with yeast genetics, followed by molecular dynamics simulations to understand the mechanisms underlying this regulation. We have identified and characterised how oligosaccharyltransferase, the central enzyme in the process of N-glycosylation, has extraordinary flexibility in selecting protein acceptor substrates due to the presence of substrate-enzyme interaction surfaces that extend far beyond the canonical requirements for glycosylation of an NxS/T "glycosylation sequon". Further, we identify a molecular switch in oligosaccharyltransferase that promotes or blocks its catalytic activity. This on/off molecular switch is controlled by binding of select protein acceptor substrates, explaining how the local sequence context of protein substrates can control the extent of site-specific N-glycosylation.