We investigated the requirement of fifteen human butyrate-producing gut bacterial strains for eight B-vitaminsand the proteinogenic amino acids by a combination of genome sequence analysis and in vitro growth experiments. The Ruminococcaceae species Faecalibacterium prausnitzii and Subdoligranulum variabile were auxotrophic for most of the vitamins and the amino acid tryptophan. Within the Lachnospiraceae, most species were prototrophic for all amino acids and most vitamins, but biotin auxotrophy was widespread. In addition, most of the strains belonging to Eubacterium rectale and Roseburia spp., but few of the other Lachnospiraceae strains, were auxotrophic for thiamine and folate. Synthetic co-culture experiments of five thiamine or folate auxotrophs with different prototrophic bacteria in the absence and presence of different vitamin concentrations revealed differences in cross-feeding efficiency between prototrophic strains. Vitamin-independent growth stimulation in co-culture compared to mono-culture was also observed, in particular for F. prausnitzii A2-165. The presence of multiple vitamin auxotrophies in the most abundant butyrate-producing Firmicutes species found in the healthy human colon indicates that these bacteria depend upon vitamins supplied from the diet, or via cross-feeding from other members of the microbial community. This is likely to be a factor in the dynamics of their populations.