Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia, a chronic and economically significant respiratory disease that affects swine production worldwide. M. hyopneumoniae adheres to, and adversely affects the function of ciliated epithelial cells of the respiratory tract, and the cilium adhesin (P97), is intricately but not exclusively involved in this process. Although binding of pathogenic bacteria to glycosaminoglycans is a recognised step in pathogenesis, knowledge of glycosaminoglycan-binding proteins is lacking in M. hyopneumoniae. However, heparin and other sulfated polysaccharides are known to block the binding of M. hyopneumoniae to purified swine respiratory cilia. In this study, four regions within the cilium adhesin were examined for their ability to bind heparin. Cilium adhesin fragments comprising 653 amino acids of the N- terminus and 301 amino acids of the C-terminus (containing two repeat regions, R1 and R2) were cloned and expressed. These fragments bound heparin in a dose-dependent and saturable manner with physiologically significant binding affinities of 0.27±0.02 µM and 1.89±0.33 µM, respectively. Heparin binding of both fragments was strongly inhibited by the sulfated polysaccharides fucoidan and mucin but not by chondroitin sulfate B. When the C-terminal repeat regions R1 and R2 were cloned separately and expressed, heparin-binding activity was lost, suggesting that both regions are required for heparin binding. The ability of the cilium adhesin to bind heparin indicates that this molecule plays a multi-functional role in the adherence of M. hyopneumoniae to host respiratory surfaces and therefore has important implications with respect to the pathogenesis of this organism.