UNLABELLED: Antimicrobial resistance is a growing public health threat worldwide that is still far from a complete understanding. Salmonella Typhimurium DT104 multiresistant strains with additional quinolone resistance are highly adaptive and have been responsible for global outbreaks and high mortality. In order to give new insights about the resistance mechanisms involved, the developed work aimed to point out subproteome changes between a DT104B clinical strain (Se20) that acquired quinolone resistance after patient treatment and an in vitro induced clonally related highly-resistant mutant (Se6-M). The intracellular subproteomes were compared by a 2-DE/LC-MS/MS approach and a total of 50 unique proteins were identified (32 more abundant in Se20 and 18 more abundant in Se6-M). The membrane subproteomes were analysed by a shotgun LC-MS/MS approach, where 7 differentially abundant proteins were identified (5 more abundant in Se6-M and 2 more abundant in Se20). Several proteins known to be directly related to quinolone resistance mechanisms (AAC(6')-Ib-cr4, OmpC, OmpD, OmpX, etc.) and MipA, recently reported as novel antibiotic resistance-related protein, were identified. Other proteins (Fur, SodA, SucB, AtpD/AtpG, OmpC, GltI, CheM/CheB, etc.) reflecting the metabolic re-adjustments occurred in each strain in order to acquire quinolone resistance were also identified. Moreover, proteins involved in lipopolysaccharide biosynthesis (RfbF, RfbG, GmhA) and export (LptA) were detected, supporting the importance of exploring these proteins as targets for the development of new antimicrobial agents. In conclusion, this study provides new insights into the mechanisms involved in the acquisition of antibiotic resistance, which can be highly valuable for the development of improved therapeutic strategies. BIOLOGICAL SIGNIFICANCE: This comparative proteomic study revealed a large number of differentially regulated proteins involved in antibiotic resistance which can be of great value to drug discovery, research and development programmes.