The development of high-throughput DNA sequencing techniques has enabled the sequencing of several hundred bacterial genomes. However, the major step towards understanding the molecular basis of an organism will be the determination of all gene functions in its genome. Current gene assignments by sequence homology generate numerous hints to putative or unknown functions. Even hits with good homology are often not specific enough to describe the in vivo biochemical functions and the underlying biological roles. In this work we applied metabolic footprinting analysis to characterize Tn916-inserted mutants of a hemicellulose-degrading rumen bacterium grown on complex culture medium. Interestingly, the most distinctive phenotypic difference was observed in a mutant with a transposon insertion in a non-coding region of the genome, while disruption of a gene with high homology to a known alpha-glucosidase/xylosidase showed no distinctive phenotypic effect. Our results demonstrate that extracellular metabolomics data coupled to genome information is a powerful and low-cost approach to rapidly screen and characterize microbial mutants with single gene deletions. However, metabolomics as a stand-alone technique is unlikely to give a complete answer to define gene functions, and, therefore, is an approach to be used to generate hypotheses and direct new experiments to confirm gene function.