Burkholderia is a ubiquitous Gram-negative genus comprised of genetically and phylogenetically diverse species. Although most members of this genus do not cause disease, some species (including B. cenocepacia, B. multivorans and B. pseudomallei) are now recognised to cause infections within immunocompromised groups and nosocomial cohorts. Previously we demonstrated the presence of an O-linked protein glycosylation system within B. cenocepacia which was required for virulence (1), yet did not define the cluster required for the generation of the O-linked glycan. Within this work, we identify the O-glycosylation (ogc) cluster necessary for synthesis, assembly, and membrane translocation of the lipid-linked O-glycan, in addition to determining the chemical structure of this trisaccharide. Surprisingly we find the ogc cluster is conserved in the Burkholderia genus and confirm the production of glycoproteins with similar glycans in B. thailandensis, B. gladioli and B. pseudomallei isolates. Further, we show that absence of protein O-glycosylation severely affects bacterial fitness and accelerates B. cenocepacia clearance in a larvae infection model. Proteomic analysis of glycosylation mutants within B. cenocepacia confirms widespread alterations within virulence associated proteins, and defects in cellular processes, supporting the importance of O-glycosylation for bacterial fitness. Finally, we demonstrate that the conservation of protein glycosylation and the ogc results in patients infected with B. cenocepacia, B. multivorans, B. pseudomallei, and B. mallei developing O-glycan specific antibodies. Together, these results highlight the importance of general protein O-glycosylation in the biology of the Burkholderia genus and its potential as a target for developing novel inhibition and immunotherapy approaches to control Burkholderia infections.