Poly‐proline II helices are secondary structure motifs frequently found in ligand binding sites. They exhibit increased flexibility and solvent exposure compared to the strongly hydrogen‐bonded α‐helices or β‐strands and can therefore easily be misinterpreted as completely unstructured regions with an extremely high rotational freedom. Here, we show that the adhesin YadA of Yersinia enterocolitica serotype O:9 contains a poly‐proline II helix interaction motif in the N‐terminal region. The motif is involved in the interaction of YadA O:9 with heparin, a host glycosaminoglycan. We show that the basic residues within the N‐terminal motif of YadA are required for electrostatic interactions with the sulphate groups of heparin. Biophysical methods including CD spectroscopy, solution‐state NMR, and SAXS all independently support the presence of a poly‐proline helix allowing YadA O:9 binding to the rigid heparin. Lastly, we show that host cells deficient in sulphation of heparin and heparan sulphate are not targeted by YadA O:9 ‐mediated adhesion. We speculate that the YadA O:9 ‐heparin interaction plays an important and highly strain‐specific role in the pathogenicity of Yersinia enterocolitica serotype O:9.