Integrity of p27-p55 operon 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, p27 and p55. p27 gene encodes a lipoprotein that binds triacylated glycolipids and modulates the host immune responses by inhibiting the MHC-II Ag processing. Besides, p55 encodes 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-p55 operon in M. bovis severely 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 p55 into the mutant strain partially restored the virulence of the bacteria while no complementation was achieved with p27 individual gene.