This work describes the identification and characterisation of a novel class of CoA-transferase involved in butyrate formation in human colonic bacteria. The CoA-transferase gene was identified form Roseburia sp. A2-183 and the gene product overexpressed in an E. coli lysates. The CoA-transferase has a broad pH optimum around neutral and shows activity with butyryl-CoA and to a lesser extent propionyl-CoA. Acetate, propionate, butyrate, isobutyrate and valerate but not 4-hydroxybutyrate, which is the preferred substrate of the closely related clostridial 4-hydroxybutyrate CoA-transferases, were used as co-substrate. The Km for butyryl-CoA and propionyl-CoA were similar but the maximal velocity of the enzyme in the presence of butyryl-CoA provided further evidence that the CoA-transferase from Roseburia sp. A2-183 is a butyryl-CoA CoA-transferase. Characterisation of the CoA-transferase substrate specificity from different butyrate-producing bacteria, including the lactate-utilising butyrate-producing Anaerostipes caccae L1-92 and Eubacterium hallii L2-7, as well as the clostridial cluster IV representative Faecalibacterium prausnitzii A2-165, indicated that the enzyme is the same in all these butyrate-producing bacteria. The metabolic cooperation between a lactic acid bacterium and the lactate-utilising butyrate-producing A. caccae L1-92 was investigated in vitro and in gnotobiotic rodents. Although the in vivo experiment failed to demonstrate the conversion of lactate to butyrate, metabolic analysis revealed significant amounts of butyrate in the caecum of di-associated animals and the establishment of a stable colonisation by the oxygen-sensitive A. caccae L1-92 represents nevertheless an essential step in assessing the potential of the butyrate-producing bacteria for probiotic application.