ABSTRACTMesomycoplasma hyopneumoniaeis the etiological agent of mycoplasmal pneumonia of swine (MPS), which causes substantial economic losses to the world’s swine industry. Moonlighting proteins are increasingly being shown to play a role in the pathogenic process ofM. hyopneumoniae. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in glycolysis, displayed a higher abundance in a highly virulent strain ofM. hyopneumoniaethan in an attenuated strain, suggesting that it may have a role in virulence. The mechanism by which GAPDH exerts its function was explored. Flow cytometry and colony blot analysis showed that GAPDH was partly displayed on the surface ofM. hyopneumoniae. Recombinant GAPDH (rGAPDH) was able to bind PK15 cells, while the adherence of a mycoplasma strain to PK15 was significantly blocked by anti-rGAPDH antibody pretreatment. In addition, rGAPDH could interact with plasminogen. The rGAPDH-bound plasminogen was demonstrated to be activated to plasmin, as proven by using a chromogenic substrate, and to further degrade the extracellular matrix (ECM). The critical site for GAPDH binding to plasminogen was K336, as demonstrated by amino acid mutation. The affinity of plasminogen for the rGAPDH C-terminal mutant (K336A) was significantly decreased according to surface plasmon resonance analysis. Collectively, our data suggested that GAPDH might be an important virulence factor that facilitates the dissemination ofM. hyopneumoniaeby hijacking host plasminogen to degrade the tissue ECM barrier.IMPORTANCEMesomycoplasma hyopneumoniaeis a specific pathogen of pigs that is the etiological agent of mycoplasmal pneumonia of swine (MPS), which is responsible for substantial economic losses to the swine industry worldwide. The pathogenicity mechanism and possible particular virulence determinants ofM. hyopneumoniaeare not yet completely elucidated. Our data suggest that GAPDH might be an important virulence factor inM. hyopneumoniaethat facilitates the dissemination ofM. hyopneumoniaeby hijacking host plasminogen to degrade the extracellular matrix (ECM) barrier. These findings will provide theoretical support and new ideas for the research and development of live-attenuated or subunit vaccines againstM. hyopneumoniae.