Borrelia burgdorferi produces Pfs and LuxS enzymes for breakdown of the toxic byproducts of methylation reactions, producing 4,5-dihydroxy-2,3-pentanedione (DPD), adenine, and homocysteine. DPD and its spontaneously rearranged derivatives constitute a class of bacterial pheromones named autoinducer-2 (AI-2). We describe that B. burgdorferi produces DPD during laboratory cultivation. Furthermore, addition of in vitro synthesized DPD to cultured B. burgdorferi resulted in altered expression levels of a specific set of bacterial proteins, among which is the outer surface lipoprotein VlsE. While a large number of bacteria utilize homocysteine, the other LuxS product, for synthesis of methionine as part of the activated-methyl cycle, B. burgdorferi was found to lack that ability. We propose that the main function of B. burgdorferi LuxS is to synthesize DPD and that the Lyme disease spirochete utilizes a form of DPD as a pheromone to control gene expression.