Shewanella oneidensis MR-1 is a gram-negative facultative aerobic bacterium living at oxic-anoxic interfaces in nature. The plasticity of terminal electron-acceptors used under anaerobic conditions is huge, but the adaptation to these different environmental conditions remains unclear. In this work, we used a proteomic approach to study the protein content when the organism is grown under anaerobic respiration conditions on insoluble ferric oxide. By analysis of two-dimensional gel patterns of soluble protein extracts, we discovered 20 differentially displayed proteins. The protein spots were further analyzed by mass spectrometry for which we used, in addition to nano-high-performance liquid chromatography coupled to an electrospray ionization-quadrupole-time of flight instrument, a recently introduced matrix-assisted laser desorption/ionization (MALDI) tandem-time of flight mass spectrometer. The instrument allows the acquisition of high quality spectra, in both the mass spectrometry and tandem mass spectrometry mode, and is therefore able to identify protein spots unambiguously. Advantageous to electrospray ionization is a minimised sample handling, inherent to MALDI ionization, and the presence of high energy fragmentation ions, generating sequence information that also can differentiate isobaric amino acids. With this strategy, we could point out a regulatory protein that is up-regulated under iron(III) respiration. This protein, the aerobic respiration control protein (ArcA), has been reported as being a regulator during anaerobiosis in other species. To our knowledge, this is the first report of the possible involvement of ArcA from S. oneidensis MR-1 in the reduction of ferric oxide.