Edited by Chris Whitfield Autoinducer-2 (AI-2) is unique among quorum-sensing signaling molecules, as it is produced and recognized by a wide variety of bacteria and thus facilitates interspecies communication. To date, two classes of AI-2 receptors have been identified: the LuxP-type, present in the Vibrionales, and the LsrB-type, found in a number of phylogenetically distinct bacterial families. Recently, AI-2 was shown to affect the colonization levels of a variety of bacteria in the microbiome of the mouse gut, including members of the genus Clostridium, but no AI-2 receptor had been identified in this genus. Here, we identify a noncanonical, functional LsrB-type receptor in Clostridium saccharobutylicum. This novel LsrB-like receptor is the first one reported with variations in the binding-site amino acid residues that interact with AI-2. The crystal structure of the C. saccharobutylicum receptor determined at 1.35 Å resolution revealed that it binds the same form of AI-2 as the other known LsrB-type receptors, and isothermal titration calorimetry (ITC) assays showed that binding of AI-2 occurs at a submicromolar concentration. Using phylogenetic analysis, we inferred that the newly identified noncanonical LsrB receptor shares a common ancestor with known LsrB receptors and that noncanonical receptors are present in bacteria from different phyla. This led us to identify putative AI-2 receptors in bacterial species in which no receptors were known, as in bacteria belonging to the Spirochaetes and Actinobacteria phyla. Thus, this work represents a significant step toward understanding how AI-2-mediated quorum sensing influences bacterial interactions in complex biological niches.