Glycosylation of flagellins is a well recognized property of many bacterial species. In this study we describe the structural characterization of novel flagellar glycans from a number of hypervirulent strains of C. difficile. We used mass spectrometry (nano LC- MS and MS/MS analysis) to identify a number of putative glycopeptides which carried a variety of glycoform substitutions each of which was linked through an initial HexNAc residue to Ser or Thr. Detailed analysis of a LLDGSSTEIR glycopeptide released by tryptic digestion, which carried two variant structures, revealed that the glycopeptide contained, in addition to carbohydrate moieties, a novel structural entity. A variety of Electrospray-MS strategies using Q-TOF technology were used to define this entity, including positive- and negative-ion collisionally activated decomposition (CAD) MS/MS which produced unique fragmentation patterns, and high resolution accurate mass measurement to allow derivation of atomic compositions, leading to the suggestion of a Taurine-containing peptidylamido-glycan structure. Finally NMR analysis of flagellin glycopeptides provided complementary information. The glycan portion of the modification was assigned as α-Fuc3N-(1→3)-α-Rha-(1→2)-α-Rha3OMe-(1→3)-β-GlcNAc-(1→)Ser and the novel capping moiety was shown to be comprised of Taurine, Alanine, and Glycine. This is the first report of a novel O-linked sulphonated peptidylamido-glycan moiety decorating a flagellin protein.