In order to survive in the host and initiate infection, Salmonella enterica needs to undergo a transition between aerobic and anaerobic growth by modulating its central metabolic pathways. In this study, a comparative analysis of the proteome of S. enterica serovar Typhimurium grown in the presence or absence of oxygen was performed. The most prominent changes in expression were measured in a semiquantitative manner using difference in-gel electrophoresis (DIGE) to reveal the main protein factors involved in the adaptive response to anaerobiosis. A total of 38 proteins were found to be induced anaerobically, while 42 were repressed. The proteins of interest were in-gel digested with trypsin and identified by MALDI TOF mass spectrometry using peptide mass fingerprinting. In the absence of oxygen, many fermentative enzymes catalysing reactions in the mixed-acid or arginine fermentations were overexpressed. In addition, the enzyme fumarate reductase, which is known to provide an alternative electron acceptor for the respiratory chains in the absence of oxygen, was shown to be induced. Increases in expression of several glycolytic and pentose phosphate pathway enzymes, as well as two malic enzymes, were detected, suggesting important roles for these in anaerobic metabolism. Substantial decreases in expression were observed for a large number of periplasmic transport proteins. The majority of these are involved in the uptake of amino acids and peptides, but permeases transporting iron, thiosulphate, glucose/ galactose, glycerol 3-phosphate and dicarboxylic acids were also repressed. Decreases in expression were also observed for a superoxide dismutase, ATP synthase, inositol monophosphatase, and several chaperone and hypothetical proteins. The changes were monitored in two different isolates, and despite their very similar expression patterns, some variability in the adaptive response to anaerobiosis was also observed.