N‐glycosylation is an important post‐translational modification of proteins that has been highly conserved during evolution and is found in Eukaryota, Bacteria and Archaea. In eukaryotes, N‐glycan processing is sequential, involving multiple specific steps within the secretory pathway as proteins travel through the endoplasmic reticulum and the Golgi apparatus. In this review, we first summarize the different steps of the N‐glycan processing and further describe recent findings regarding the diversity of N‐glycan structures in eukaryotic clades. This comparison allows us to explore the different regulation mechanisms of N‐glycan processing among eukaryotic clades. Recent findings regarding the regulation of protein N‐glycosylation are highlighted, especially the regulation of the biosynthesis of complex‐type N‐glycans through manganese and calcium homeostasis and the specific role of transmembrane protein 165 (TMEM165) for which homologous sequences have been identified in several eukaryotic clades. Further research will be required to characterize the function of TMEM165 homologous sequences in different eukaryotic clades.