Toxigenic Clostridium difficile strains produce toxin A and toxin B, which have been implicated in pathogenesis; however, the molecular mechanisms regulating toxin production are elusive. Darkoh et al. now show that the Agr quorum sensing system has a role in toxin expression through the generation of a novel thiolactone signalling peptide. Purified fractions containing this peptide from C. difficile cultures and stool samples of infected patients stimulated toxin synthesis in both hypervirulent and non-hypervirulent C. difficile strains. In addition to the newly identified signalling peptide, components of the quorum sensing and response pathways were shown to be involved in toxin expression. Thus, this quorum signalling pathway may provide new avenues for the development of therapeutics that target C. difficile.