Pelotomaculum thermopropionicum is a syntrophic propionate-oxidizing bacterium that catalyses the intermediate bottleneck step of the anaerobic-biodegradation process. As it thrives on a very small energy conserved by propionate oxidation under syntrophic association with a methanogen, its catabolic pathways and regulatory mechanisms are of biological interest. In this study, we constructed high-density oligonucleotide microarrays for P. thermopropionicum, and used them to analyse global transcriptional responses of this organism to different growth substrates (propionate, ethanol, propanol and lactate) in co-culture with a hydrogenotrophic methanogenic archaeon, Methanothermobacter thermautotrophicus (by reference to fumarate monoculture). We found that a substantial number of genes were upregulated in the syntrophic co-cultures irrespective of growth substrates (including those related to amino-acid and cofactor metabolism), suggesting that these processes were influenced by the syntrophic partner. Expression of the central catabolic pathway (the propionate-oxidizing methylmalonyl-CoA pathway) was found to be substrate-dependent and was largely stimulated when P. thermopropionicum was grown on propionate and lactate. This finding was supported by results of growth tests, revealing that syntrophic propionate oxidation was largely accelerated by supplementation with lactate. These results revealed that P. thermopropionicum has complex regulatory mechanisms that alter its metabolism in response to the syntrophic partner and growth substrates.