N-glycosylation plays a fundamental role in many biological processes. Human diamine oxidase (hDAO), required for hista-mine catabolism, has multiple N-glycosylation sites, but their roles, for example in DAO secretion, are unclear. We recently reported that the N-glycosylation sites Asn168, Asn538, and Asn745 in recombinant hDAO (rhDAO) carry complex-type glycans, where-as Asn110 carries only mammalian-atypical oligomannosidic glycans. Here, we show that Asn110 in native hDAO from amniotic fluid and Caco-2 cells, DAO from porcine kidneys, and rhDAO produced in two different HEK293 cell lines is also consistently occu-pied by oligomannosidic glycans. Glycans at Asn168 were predominantly sialylated with bi- to tetra-antennary branches, and Asn538 and Asn745 had similar complex-type gly-cans with some tissue- and cell line-specific variation. The related copper-containing amine oxidase human vascular adhesion pro-tein-1 also exclusively displayed high-mannose glycosylation at Asn137. X-ray structures revealed that the residues adjacent to Asn110 and Asn137 form a highly con-served hydrophobic cleft interacting with the core tri-saccharide. Asn110 replacement with Gln completely abrogated rhDAO secretion and caused retention in the endoplasmic re-ticulum. Mutations of Asn168, Asn538, and Asn745 reduced rhDAO secretion by 13%, 71%, and 32%, respectively. Asn538/745 double and Asn168/538/745 triple substitu-tions reduced rhDAO expression by 85% and 94%. Because of their locations in the DAO structure, Asn538 and Asn745 glycosylations might be important for efficient DAO dimer formation. These functional results are re-flected in the high evolutionary conservation of all four glycosylation sites. Human DAO is abundant only in the gastrointestinal tract, kidney and placenta, and glycosylation seems essential for reaching high enzyme expression levels in these tissues.