Technical Abstract: The complete genome of Campylobacter jejuni strain RM1221 (Penner serotype HS:53) was reported recently and contains a novel capsular polysaccharide (CPS) biosynthesis locus. Cell surface carbohydrates such as CPS are known to be important for bacterial survival and often contribute to pathogenesis. In this study, we describe the complete structure of the CPS of C. jejuni RM1221, determined using NMR, MS, and chemical methods. The CPS contained 6-deoxy-D-manno-heptose and D-threo-pent-2-ulose (D-xylulose), two monosaccharides rarely found in bacterial polysaccharides. The CPS had a regular structure of a linear main chain of trisaccharide repeating units, composed of two '- and one '- 6-deoxy-D-manno-heptopyranose residues, linked through a phosphodiester linkage. Branching residues of xylulose were incorporated non-stoichiometrically: each trisaccharide repeating unit of the main chain beared none, one or two xylulose residues. The xylulose glycosidic linkages are extremely acid labile, and it is not clear how they can be preserved in acidic conditions of gastrointestinal tract, where Campylobacter resides during infection. We have also showed that the CPS biosynthesis genes of C. jejuni RM1221 are conserved in other C. jejuni strains of the Penner serotype HS:53 , including serotype HS:53 reference strain RM3435.