For microorganisms that play an important role in bioremediation, the adaptation to swift changes in the availability of various substrates is a key for survival. The iron-reducing bacterium Geobacter metallireducens was hypothesized to repress utilization of less preferred substrates in the presence of high concentrations of easily degradable compounds. In our experiments, acetate and ethanol were preferred over benzoate, but benzoate was co-consumed with toluene and butyrate. To reveal overall physiological changes caused by different single substrates and a mixture of acetate plus benzoate, a nano-liquid chromatography-tandem mass spectrometry-based proteomic approach (nano-LC-MS/MS) was performed using label-free quantification. Significant differential expression during growth on different substrates was observed for 155 out of 1477 proteins. The benzoyl-CoA pathway was found to be subjected to incomplete repression during exponential growth on acetate in the presence of benzoate and on butyrate as a single substrate. Peripheral pathways of toluene, ethanol, and butyrate degradation were highly expressed only during growth on the corresponding substrates. However, low expression of these pathways was detected in all other tested conditions. Therefore, G. metallireducens seems to lack strong carbon catabolite repression under high substrate concentrations, which might be advantageous for survival in habitats rich in fatty acids and aromatic hydrocarbons. © 2014 Elsevier GmbH.