Mycoplasma bovis is one of the serious pathogens causing bovine respiratory diseases. Meanwhile,M. bovis can cause a variety of clinical signs,including bronchopneumonia,mastitis,arthritis,genital tract inflammation,and tenosynovitis. Adhesion to airway epithelial cells is a key step for M. bovis colonization and invasion of cells. Adhesin is one of the main virulence factors of M. bovis. Research about attachment has become an important field of pathogenic mechanisms research of M. bovis. To further analyze the adhesion characteristics of the M. bovis VspX protein,experiments,including indirect immunofluorescence assay,adhesion assay of protein and mutant strain to embryonic bovine lung (EBL) cells,antibody inhibition adhesion detection,ELISA assay of protein and strains binding fibronectin (Fn),were carried out,to clarify the molecular mechanism of VspX protein adhesion. The results showed that the M. bovis VspX protein was located on the surface of the strain. The recombinant VspX protein (rVspX) was able to adhere to the embryonic bovine lung (EBL) cells. Compared with the M. bovis wild strain (M. bovis WT),the M. bovis VspX gene-deleted mutant strain (M. bovis ΔVspX) had a significantly decreased ability to adhere to EBL cells in vitro (P<0.05). The above results showed that the VspX protein was an adhesion-related protein. The anti-rVspX monoclonal antibody was able to inhibit the adhesion of M. bovis to EBL cells,which further confirmed the specificity of the adhesion. Also,the rVspX protein bound to Fn in a dose-dependent manner,and the M. bovis ΔVspX strain had a significantly lower adherence ability to Fn (P<0.05) than that of the M. bovis WT strain. These results further demonstrated the binding specificity of M. bovis VspX protein to Fn. And Fn was distributed on the surface of EBL cells. In summary,the study validated that M. bovis VspX protein was an adhesion-related protein with Fn binding properties,and the adhesion of VspX to EBL cells was mediated by the extracellular matrix component,Fn.