Campylobacter jejuni is the most common cause of food-borne illness worldwide, accounting for ~225,000 infections in Australia per year. Infection is zoonotic and is associated with consumption of under-cooked or poorly prepared poultry, in which C. jejuni is an asymptomatic commensal. A unique molecular feature of C. jejuni is the ability to post-translationally modify membrane-associated proteins by the N-linked addition of a heptasaccharide glycan. Glycan biosynthesis is performed by proteins encoded within the pgl (protein glycosylation) locus, and attachment is mediated by the PglB oligosaccharyltransferase. Disruption of pgl genes reduces chicken colonization, adhesion to, and invasion of, human epithelial cells, however the proteins that mediate these phenotypes, and the overall function of the N-glycan, remain to be determined. We developed mass spectrometric techniques that have allowed the identification of >120 sites of N-linked glycosylation in C. jejuni, and identified a non-canonical glycan that is itself modified by addition of phosphoethanolamine (pEtN) to terminal GalNAc. Quantitative proteomics has shown elevated abundance of Pgl proteins under conditions that mimic host environments (e.g. deoxycholate, low iron, high salt), in chicken exudate (‘juice’), and in clinical rather than laboratory-adapted strains, further confirming a role for glycosylation in host colonization. Increased Pgl protein expression leads to elevated N-glycan biosynthesis and attachment that targets specific proteins. Quantitative glycoproteomics also revealed glycopeptides that lack the typical C. jejuni glycosylation sequon (D/E-X-N-X-S/T). Glycan-deficient mutants are incapable of interacting with almost all human-type cell surface glycans and infection of epithelial cell models with C. jejuni depletes complex-type cell surface glycans, indicating glycan-glycan interactions are necessary for host colonization. We are also exploring the function of the N-glycan in resistance against proteolytic activity in the C. jejuni periplasm and in the formation of membrane-associated protein complexes.