Mycoplasma bovis, a minimal bacterium but a notorious cattle pathogen, leads to serious economic losses. This pathogen binding to host cells is emerging as a complex process involving a broad range of surface-exposed structures. For mycoplasma, adhering to host tissue is the first and crucial step of infection. It is well known that the molecules contributing to microbial cytoadhesion are important virulence factors. Here, we cloned, expressed, and purified the recombinant protein, which is encoded by MBOVJF4278_00820, and induced polyclonal antibodies for it in mice. The cytoadhesive properties of this surface-exposed protein were demonstrated on embryonic bovine lung (EBL) and Madin-Darby bovine kidney (MDBK) cells. Furthermore, heparin as the binding target was confirmed, and the characteristics of the interaction between this protein and heparin have also been analyzed. Our data indicate that the surface-associated MBOVJF4278_00820-encoded protein is a novel adhesion-related factor of Mycoplasma bovis.IMPORTANCEAdhesins are crucial in facilitating Mycoplasma bovis infection. In this study, we identified a specific Mycoplasma bovis adhesin that interacts with heparin on the surface of host cells. Given that heparin is ubiquitously distributed across a wide range of tissue cells, the identification of the heparin-binding adhesin is significant for elucidating how Mycoplasma bovis targets diverse host cells and triggers a spectrum of clinical manifestations.