The Gram-negative, flagellated, anaerobic, crescent-shaped bacterium Selenomonas sputigena is a potential human periodontal pathogen. Information on its virulence factors and underlying pathogenicity mechanisms is scarce. Here we show for the first time that S. sputigena produces a diversely and heavily O-glycosylated flagellin C9LY14 as a major cellular protein, which carries various hitherto undescribed rhamnose- and N-acetylglucosamine linked O-glycans in the range from mono- to hexasaccharides. A comprehensive glycomic and glycoproteomic assessment revealed extensive glycan macro- and microheterogeneity identified from 22 unique glycopeptide species. From the multiple sites of glycosylation, five were unambiguously identified on the 437-amino acid C9LY14 protein (Thr149, Ser182, Thr199, Thr259, and Ser334), the only flagellin protein identified. The O-glycans additionally showed modifications by methylation and putative acetylation. Some O-glycans also carried hitherto undescribed residues/modifications as determined by their respective m/z values, reflecting the high diversity of native S. sputigena flagellin. We also found that monosaccharide rearrangement occurred during collision-induced dissociation (CID) of protonated glycopeptide ions. This effect resulted in pseudo Y1-glycopeptide fragment ions that indicated the presence of additional glycosylation sites on a single glycopeptide. CID oxonium ions and electron transfer dissociation, however, confirmed that just a single site was glycosylated, domonstrating for the first time that glycan to peptide rearrangement can occur on glycopeptides and that this effect is influenced by the molecular nature of the glycan moiety. This effect was most pronounced with disaccharides. This study is the first report on O-linked flagellin glycosylation in S. sputigena, revealing that C9LY14 is one of the most heavily glycosylated flagellins described to date. The data have been deposited to the ProteomeXchange with identifier PXD005859