The Cronobacter spp., previously known as Enterobacter sakazakii, are Gram-negative enterobacterial pathogens that can cause necrotizing enterocolitis, meningitis, and septicemia with a high mortality rate in neonates. The O-specific polysaccharide (O-PS) was isolated from Cronobacter sakazakii strain 767 and structurally characterized using (1)H and (13)C NMR spectroscopy, including two-dimensional DQF-COSY, TOCSY, ROESY, HSQC, and HMBC experiments. Further compositional determination was undertaken using classical chemical methods followed by GLC, and GLC-MS analysis. The repeating unit of O-PS isolated from C. sakazakii 767 was a branched heptasaccharide composed of l-Rha, d-Glc, d-GlcNAc, and d-GalA, and had the structure shown below. One of the Rha residues was partially O-acetylated at C-4. C. sakazakii 767 was originally isolated from a fatal neonatal meningitic case, and the structure of its O-PS significantly differs from the O-PS structures previously described for Cronobacter spp.