Brucellosis is a zoonotic infection caused by bacteria of the genus Brucella. The species, B. abortus and B. melitensis, major causative agents of human brucellosis, share remarkably similar genomes, but they differ in their natural hosts, phenotype, antigenic, immunogenic, proteomic and metabolomic properties. In the present study, label-free quantitative proteomic analysis was applied to investigate protein expression level differences. Type strains and field strains were each cultured six times, cells were harvested at a midlogarithmic growth phase and proteins were extracted. Following trypsin digestion, the peptides were desalted, separated by reverse-phase nanoLC, ionized using electrospray ionization and transferred into an linear trap quadrapole (LTQ) Orbitrap Velos mass spectrometer to record full scan MS spectra (m/z 300–1700) and tandem mass spectrometry (MS/MS) spectra of the 20 most intense ions. Database matching with the reference proteomes resulted in the identification of 826 proteins. The Cluster of Gene Ontologies of the identified proteins revealed differences in bimolecular transport and protein synthesis mechanisms between these two strains. Among several other proteins, antifreeze proteins, Omp10, superoxide dismutase and 30S ribosomal protein S14 were predicted as potential virulence factors among the proteins differentially expressed. All mass spectrometry data are available via ProteomeXchange with identifier PXD006348.