Rhodococcus equi is an intracellular pathogen which harbours a virulence plasmid that is essential for inhibiting the maturation of the endosome in which it resides, thus preventing it from being killed by the host cell. R. equi isolates from different animal hosts harbour different types of the virulence plasmid. The vapA type virulence plasmid (pVAPA) is typically identified in equine isolates, the vapB type virulence plasmid (pVAPB) occurs in porcine isolates, while the vapN type virulence plasmid (pVAPN) was recently identified in bovine isolates. VapA is the key virulence factor encoded within pathogenicity island (PAI) in pVAPA. In this study, macrophage infection approach was employed to investigate the functionality of Vaps in three types of virulence plasmid. vapA was the only vap gene in pVAPA essential for virulence in equine isolates. Only vapK (K1/K2) in pVAPB and vapN in pVAPN have the ability to compensate the virulence of vapA in equine isolates with the deletion of vapA. We found that only one vap gene in each type of virulence plasmid was essential for virulence.