Protein N-glycosylation is a post-translational modification that exists in all domains of life. It consists of two main steps : the biosynthesis of a lipid-linked oligosaccharide (LLO), and the glycan transfer reaction from LLO to asparagine residues of acceptor proteins, a process catalyzed by oligosaccharyltransferase (OST) enzymes. Over the past few years, my research has focused on structural and mechanistic investigation of N-glycosylation enzymes using a multidisciplinary approach that involves protein biochemistry, chemo-enzymatic glycan elongation, X-ray crystallography, and single-particle Cryo-EM. We described a counting mechanism for PglH, a processive glycosyltransferase involved in LLO biosynthesis in the bacterial human pathogen Campylobacter jejuni. In higher eukaryotes, where OST enzymes are multimeric membrane complexes located at the membrane of the endoplasmic reticulum (ER), we determined cryo-EM structures of the two OST enzymes that co-exist in human cells, revealing the molecular basis of their specific function in catalyzing co-translational or post-translational N-glycosylation. Recently, we elucidated cryo-EM structures of the yeast multimeric OST complex in different functional states revealing the molecular basis of substrate recognition and catalysis.