Vibrio anguillarum is a Gram-negative bacterium associated with vibriosis in Atlantic cod (Gadus morhua L.). Although farmed cod in Norway is routinely vaccinated against the infection, outbreaks of V. anguillarum-associated vibriosis still occur. Here, we describe the structural characterization of the LPS O-chain polysaccharide (O-PS) from atypical isolates of V. anguillarum strain 1282 and show that it is distinct from that previously established for V. anguillarum serotype O2. The structure of the purified O-PS was shown by 1D/2D NMR ((1)H, (13)C) spectroscopy and CE-MS studies to be a high-molecular mass linear polymer of tetrasaccharide repeating units, composed of 2-acetamido-3-(N-formyl-L-alanyl)amido-2,3-dideoxy-D-glucuronamide [GlcNAc3N(Fo-L-Ala)AN], 2-acetamido-3-acetamidino-2,3-dideoxy-D-mannuronic acid (ManNAc3NAmA), 3-acetamido-3-dideoxy-D-quinovose (Qui3NAc), and 2,4-diacetamido-2,4-dideoxy-D-fucose (FucNAc4NAc). [carbohydrate structure: see text]. NMR analysis of the partial hydrolysis-derived oligosaccharides confirmed the presence of an O-acetyl group at position O-4 of GlcNAc3N(Fo-L-Ala)AN and established that the above-mentioned structure represents the biological repeating unit of the O-PS. In addition, it was demonstrated that some of 2,3-diamino-2,3-dideoxy-glucuronamide in the O-PS was present in the form of 2,3-diamino-2,3-dideoxy-glucose.