Burkholderia cenocepacia is a leading cause of necrotizing pneumonia in cystic fibrosis patients worldwide. In addition to numerous characterized virulence factors, B. cenocepacia possesses a general O-linked glycosylation required for pathogenesis in model systems, yet how O-linked glycosylation influences virulence is unknown. Within this opportunistic pathogen, O-linked protein glycosylation is mediated by two non-linked genetic components; the O-oligosaccharyltransferase (PglL) and the O-glycan cluster (OGC), which are both conversed across Burkholderia species. Using proteomic based approaches, we demonstrate that the loss of O-linked protein glycosylation in ∆PglL, ∆OGC and ∆PglL∆OGC strains leads to multiple changes in known virulence factors such as the flagella apparatus and in transcriptional control/DNA-binding factors. Surprisingly, these observed changes are consistent with inhibition of the known quorum sensing systems cepIR. DNA cross-linking proteomic studies and luciferase assays supports the loss of glycosylation, leading to an altered DNA binding landscape. Analysis of known O-linked glycoproteins demonstrates that only a single glycoprotein is effected by the loss of O-linked glycosylation, a putative lipoprotein we have termed glycosylation sensitive lipoprotein (GSL). Degradomics analysis supports that GSL and other glycoproteins are processed within the periplasm, with the loss of glycosylation promoting protein processing around glycosylation sites. Thus, these studies support a model where the loss of O-linked glycosylation leads to changes in cepIR regulation due to alteration in only in a subset of B. cenocepacia glycoproteins. Using proteomic and degradomics approaches we identify a single protein, GSL, which may be responsible for driving the attenuation observed in glycosylation null B. cenocepacia strains.