The structure of the tetrameric Vicia villosa isolectin B4 (VVLB4) in complex with a cancer antigen, the Tn glycopeptide (GalNAc-O-Ser), was determined at 2.7 A resolution. The N-acetylgalactoside moiety of the ligand binds to the primary combining site of VVLB4 in a similar way as observed for other Gal/GalNAc-specific plant lectins. The amino acid moiety of the Tn antigen is largely exposed to the solvent and makes few contacts with the protein. The structure of the complex provides a framework to understand the differences in the strength of VVLB4 binding to different sugars and emphasizes the role of a single protein residue, Tyr127, as a structural determinant of Tn-binding specificity.