The ability of rhizobia to symbiotically fix nitrogen from the atmosphere when forming nodules on their plant hosts requires various signal transduction pathways. LuxR-LuxI-type quorum-sensing systems have been shown to be one of the players in a number of rhizobium species. In this study, we found that Mesorhizobium tianshanense, a moderate-growth Rhizobium that forms nodules on a number of licorice plants, produces multiple N-acyl homoserine lactone (AHL)-like molecules. A simple screen for AHL synthase genes using an M. tianshanense genomic expression library in Escherichia coli, coupled with a sensitive AHL detector, uncovered a LuxI-type synthase, MrtI, and a LuxR-type regulator, MrtR, in M. tianshanense. Deletions of the mrtI or mrtR locus completely abolished AHL production in M. tianshanense. Using lacZ transcriptional fusions, we found that expression of the quorum-sensing regulators is autoinduced, as mrtI gene expression requires MrtR and cognate AHLs and mrtR expression is dependent on AHLs. Compared with the wild-type strains, quorum-sensing-deficient mutants showed a marked reduction in the efficiency of root hair adherence and, more importantly, were defective in nodule formation on their host plant, Glycyrrhiza uralensis. These data provide strong evidence that quorum sensing plays a critical role in the M. tianshanense symbiotic process.