AbstractThe Riemerella anatipestifer bacterium is known to cause infectious serositis in ducklings. Moreover, its adherence to the host’s respiratory mucosa is a critical step in pathogenesis. Membrane cofactor protein (MCP; CD46) is a complement regulatory factor on the surface of eukaryotic cell membranes. Bacteria have been found to bind to this protein on host cells. Outer membrane proteins (OMPs) are necessary for adhesion, colonisation, and pathogenicity of Gram-negative bacteria; however, the mechanism by which R. anatipestifer adheres to duck cells remains unclear. In this study, pull-down assays and LC–MS/MS identified eleven OMPs interacting with duck CD46 (dCD46), with OMP71 exhibiting the strongest binding. The ability of an omp71 gene deletion strain to bind dCD46 is weaker than that of the wild-type strain, suggesting that this interaction is important. Further evidence of this interaction was obtained by synthesising OMP71 using an Escherichia coli recombinant protein expression system. Adhesion and invasion assays and protein and antibody blocking assays confirmed that OMP71 promoted the R. anatipestifer YM strain (RA-YM) adhesion to duck embryo fibroblasts (DEFs) by binding to CD46. Tests of the pathogenicity of a Δomp71 mutant strain of RA-YM on ducks compared to the wild-type parent supported the hypothesis that OMP71 was a key virulence factor of RA-YM. In summary, the finding that R. anatipestifer exploits CD46 to bind to host cells via OMP71 increases our understanding of the molecular mechanism of R. anatipestifer invasion. The finding suggests potential targets for preventing and treating diseases related to R. anatipestifer infection.