Significance This article describes the characterization of the antigenic determinant of the capsular polysaccharide from the clinically relevant serotype III of group B Streptococcus (GBS). NMR and X-ray crystallography have been applied to elucidate the interaction of type III GBS oligosaccharides obtained by synthetic and depolymerization procedures of the bacterial polysaccharide with a functional monoclonal antibody. A Fab–GBS oligosaccharide complex structure has been solved at high resolution (2.7 Å). The results demonstrate the existence of a sialic acid-dependent functional epitope of GBS that is fully contained within four consecutive sugars deriving from the type III GBS polysaccharide backbone and one branched disaccharide present in this sequence. This finding has implications for the development of vaccines against GBS infection.