Mycoplasma hyopneumoniae, the causative agent of porcine enzootic pneumonia, colonises the respiratory cilia of affected swine causing significant economic losses to swine production worldwide. Heparin is known to inhibit adherence of M. hyopneumoniae to porcine epithelial cilia. M. hyopneumoniae cells bind heparin but the identity of the heparin-binding proteins is limited. Proteomic analysis of M. hyopneumoniae lysates identified 27 kDa (P27), 110 kDa (P110) and 52 kDa (P52) proteins representing different regions of a 159 kDa (P159) protein derived from mhp494. These cleavage fragments were surface located and present at all growth stages. Following purification of 4 recombinant proteins spanning P159 (F1P159, F2P159, F3P159, and F4P159), only F3P159 and F4P159 bound heparin in a dose-dependent manner (Kd values 142.37 + 22.01 nM; 75.37 + 7.34 nM respectively). Scanning electron microscopic studies showed M. hyopneumoniae bound intimately to porcine kidney epithelial-like cells (PK15 cells) but these processes were inhibited by excess heparin and F4P159. Similarly, latex beads coated with F2P159 and F4P159 adhered to and entered PK15 cells, but heparin, F2P159, and F4P159 was inhibitory. These findings indicate that P159 is a post-translationally cleaved, glycosaminoglycan-binding adhesin of M. hyopneumoniae.