Multidrug-resistant Acinetobacter baumannii strains have been examined at the DNA sequence level, but seldom using large-scale quantitative proteomics. We have compared the proteome of the multidrug resistant strain BAA-1605, with the proteome of the drug-sensitive strain ATCC 17978, using iTRAQ labeling and online 2D LC/MS/MS for peptide/protein identification. Of 1484 proteins present in at least 2 of 4 independent experiments, 114 are 2-fold to 66-fold more abundant in BAA-1605, and 99 are 2-fold to 50-fold less abundant. Proteins with 2-fold or greater abundance in the multidrug resistant strain include drug-, antibiotic-, and heavy metal-resistance proteins, stress-related proteins, porins, membrane transporters, proteins important for acquisition of foreign DNA, biofilm-related proteins, cell-wall and exopolysaccharide-related proteins, lipoproteins, metabolic proteins, and many with no annotated function. The porin CarO, inactivated in carbapenem-resistant strains, is 2.3-fold more abundant in BAA-1605. Likewise, the porin OmpW, less abundant in carbapenem- and colistin-resistant A. baumannii strains, is 3-fold more abundant in BAA-1605. Nine proteins, all present in the drug-sensitive strain but from 2.2-fold to 16-fold more abundant in the MDR strain, can potentially account for the observed resistance of BAA-1605 to 18 antibiotics. BIOLOGICAL SIGNIFICANCE: Multidrug resistant (MDR) strains of the pathogen Acinetobacter baumannii are a significant cause of hospital-acquired infections, are associated with increased mortality and length of stay, and may be a major factor underlying the spread of this pathogen, which is difficult to eradicate from clinical settings. To obtain a better understanding of antimicrobial resistance mechanisms in MDR A. baumannii, we report the first large scale 2D LC/MS/MS-based quantitative proteomics comparison of a drug-sensitive strain and an MDR strain of this pathogen. Ca. 20% of the expressed proteome changes 2-fold or more between the compared strains, including 42 proteins with literature or informatics annotations related to resistance mechanisms, modification of xenobiotics, or drug transport. Other categories of proteins differing 2-fold or more between strains include stress-response related proteins, porins, OMPs, transporters and secretion-related proteins, cell wall- and expolysaccharide-related proteins, lipoproteins, and DNA- and plasmid-related proteins. While the compared strains also differ in other aspects than multi-drug resistance, the observed differences, combined with protein functional annotation, suggest that complex protein expression changes may accompany the MDR phenotype. Expression changes of nine proteins in the MDR strain can potentially account for the observed resistance to 18 antibiotics.