Clostridium difficile, a Gram-positive spore-forming anaerobe, causes infections inhumans ranging from mild diarrhoeal to potentially life-threatening pseudomembranous colitis. The availability of genomic information for a range of C. difficile strains affords researchers the opportunity to better understand not only theevolution of these organisms but also their basic physiology and biochemistry. We used proteomics to characterize the insoluble subproteome of C. difficile strain 630. Gel-based LC-MS analysis led to the identification of 2298 peptides; PROVALT analysis with a false discovery rate set at 1% concatenated this list to 560 unique peptides, resulting in 107 proteins being positively identified. These were functionally classified and physiochemically characterized and pathway reconstruction identified a variety of central anaerobic metabolic pathways, including glycolysis, mixed acid fermentation and short-chain fatty acid metabolism. Additionally, the metabolism of a variety of amino acids was apparent, including the reductive branch of the leucine fermentation pathway, from which we identified seven of the eight enzymes. Increasing proteomics data sets should – in conjunction with other‘omic’ technologies – allow the construction of models for ‘normal’ metabolism in C. difficile 630. This would be a significant initial step towards a full systems understanding of this clinically important microorganism.