Clostridium difficile is a nosocomial pathogen that can cause severe gastrointestinal infections. C. difficile encodes a family of cell wall proteins, some of which are implicated in pathogenesis. Here we have characterized CwpV, the largest member of this family. CwpV is surface expressed and post-translationally processed in a manner analogous to the major S-layer protein SlpA. Expression of cwpV is phase variable, with approximately 5% of cells in a population expressing the protein under standard laboratory growth conditions. Upstream of cwpV, inverted repeats flank a 195 bp sequence which undergoes DNA inversion. Use of a gusA transcriptional reporter demonstrated that phase variation is mediated by DNA inversion; in one orientation cwpV is expressed while in the opposite orientation the gene is silent. The inversion region contains neither the promoter nor any of the open reading frame, therefore this system differs from previously described phase variation mechanisms. The cwpV promoter is located upstream of the inversion region and we propose a model of phase variation based on intrinsic terminator formation in the OFF transcript. A C. difficile site-specific recombinase able to catalyse the inversion has been identified.