Brucellosis is a zoonosis of the Mediterranean and Middle-East regions linked to important economic losses and reduced animal welfare. To date, no effective diagnostic and/or prophylactic measures are available for the control of brucellosis, due to the residual virulence of the bacterial strain administered for vaccinal purposes and the difficulties in distinguishing vaccinated from infected animals. To overcome these issues, studies are desired to elucidate the bacterial biology and the pathogenetic mechanisms of both the vaccinal strain and the pathogenic strains. Here, we employed a label-free shotgun proteomics to investigate the protein repertoire of the vaccinal strain B. melitensis Rev.1 and compare it with the proteome of the Brucella melitensis 16 M, one of the most common field strains isolated from ruminants. Comparative proteomics profiling underlines common and diverging traits between the two strains, providing suggestions on the potential biochemical routes responsible of the residual virulence of the vaccinal strain; whilst the diverging traits are suggestive biochemical signatures to be further investigated to provide an optimized diagnostics capable of discriminating the vaccinated from infected animals.