Clostridia coordinate many important processes such as toxin production, infection, and survival by density-dependent communication (quorum sensing) using autoinducing peptides (AIPs). Although clostridial AIPs have been proposed to be (thio)lactone-containing peptides, their true structures have remained elusive. Here, we report the genome-guided discovery of a cyclic AIP (cAIP) that controls endospore formation in Ruminiclostridium cellulolyticum . Through a combination of chemical synthesis and chemical complementation assays with mutant strain, we reveal that a homodetic cyclopeptide is the genuine chemical mediator. Kinetic analyses indicate that the mature cAIP is produced via a cryptic thiolactone intermediate that undergoes a rapid S → N shift, in a manner similar to intramolecular native chemical ligation (NCL). Finally, by implementing a chemical probe in a targeted screen, we show that this novel enzyme-primed, intramolecular NCL is a widespread feature of clostridial cAIPs.