Integrity of p27-p55operon has been demonstrated to be crucial for replication of Mycobacterium tuberculosis, the main agent of human tuberculosis, in the mouse model of infection. However, the individual contribution of each gene of the operon for the virulence of pathogenic Mycobacterium spp. still remains unclear. The operon is formed by two genes, p27and p55. p27gene encodes a lipoprotein that binds triacylated glycolipids and modulates the host immune responses by inhibiting the MHC-II Ag processing. Besides, p55encodes an efflux pump that, together with P27, is involved in resistance to drugs. In this study, we evaluated the individual contribution of P27 and P55 to the virulence of Mycobacterium bovis, the etiological agent for bovine tuberculosis.Knockout mutation of p27-p55operon in M. bovisseverely decreased the virulence of the bacteria when assessed in a progressive model of pulmonary tuberculosis in Balb/c mice. In addition, the mutant strain showed poor replication in a murine macrophagic cell line. Virulence and intracellular replication were only restored when the mutant strain was complemented with a copy of the whole operon. The reintroduction of p55into the mutant strain partially restored the virulence of the bacteria while no complementation was achieved with p27individual gene.