Clostridium difficile is a Gram positive, spore forming anaerobe and is the leading cause of hospital acquired diarrhoea. During the early 2000’s, epidemic outbreaks of C. difficile infection (CDI) were observed worldwide and these strains were found to cause more severe disease than historical strains. The diseases caused by C. difficile are toxin mediated, primarily by the two large clostridial glucosylating toxins, toxin A (TcdA) and toxin B (TcdB). Some C. difficile strains, in particular the epidemic isolates, produce an additional toxin known as C. difficile binary toxin or C. difficile transferase (CDT). CDT is composed of an enzymatic ADP-ribosyltransferase subunit (CDTa) and a receptor binding (CDTb) subunit; the structural genes for these proteins are encoded within a pathogenicity locus known as the CdtLoc. A third gene (cdtR) is located within the CdtLoc, and encodes an orphan response regulator from a two-component signal transduction system. It was previously shown that in trans CdtR increases the production of CDT in an appropriate genetic background. We have constructed independent cdtR mutants in an epidemic C. difficile isolate, M7404, by insertional inactivation using TargeTron technology. These mutants were found to produce less CDT compared to the wild type. Complementation of the cdtR mutations resulted in increased in CDT production, to a level greater than that of the wild-type strain. Overall, these results show that CdtR positively regulates CDT production in a clinically relevant epidemic C. difficile isolate.