This research habilitation focuses on the characterization of a class of enzymes - glycosyltransferases - that are involved in the biosynthesis of glycoproteins. It emphasizes the use of cryo-electron microscopy (cryo-EM) to study these enzymes, for which obtaining mechanistic insights remains a challenging task to this day. The presentation will provide an overview of the different types of glycosylation found in humans, along with a detailed description of the biosynthetic pathways of N-linked glycans and glycosaminoglycans. Subsequently, I describe my work over the past ten years, which is divided into two parts : my postdoctoral studies on a central enzyme complex of the N-linked glycosylation machinery and the work of my research team at the Institut de Biologie Structurale focusing on heparan sulfate and chondroitin sulfate biosynthesis. It closes with an overview on ongoing and future projects.​