Many Gram-negative pathogens use N-acylhomoserine lactones as signaling molecules, whereas some Gram-positive bacteria use species-specific oligopeptides. A third cell-to-cell signal molecule is autoinducer-2, produced by a variety of Gram-negative and Gram-positive bacteria. AI-2 is therefore often called an interspecies signaling molecule. A few well-known pathogens, including Vibrio spp. and Salmonella, use AI-2 as a cue to sense population density. The key enzyme for AI-2 production is LuxS, which is an essential part of the activated methyl cycle, involved in recycling S-adenosylhomocysteine. More specifically, LuxS catalyzes the cleavage of S-ribosyl-homocysteine to homocysteine and 4,5-dihydroxy-2,3-pentanedione, which subsequently leads to the production of AI-2. A wide range of bacterial species produce AI-2, but evidence for the presence of signal reception and signal transduction pathways in organisms besides Escherichia coli, Vibrio spp. and Salmonella is lacking. While this lack of evidence is sometimes used to question the role of AI-2 in interspecies signaling, an alternative explanation is that other types of receptors and signal transduction pathways are yet to be discovered. Although the interspecies signaling molecule AI-2 is commonly linked to virulence and pathogenicity, it has recently been shown that the probiotic strain Lactobacillus acidophilus NCFM harbors a functional luxS gene and produces AI-2. Whether this signaling molecule plays a role in eliciting the beneficial traits of probiotic bacteria remains to be determined. Indeed, it was suggested that the ability to produce AI-2 affects attachment of L. acidophilus to intestinal epithelial cells, as a mutation in luxS was shown to result in decreased adherence to Caco-2 cells. Additionally, luxS has been attributed a central metabolic role in Lactobacillus reuteri 100–23 and Lactobacillus rhamnosus GG, and has been shown to influence adherence, biofilm formation and exopolysaccharide production in the latter. In a recent study AI-2 production has been demonstrated for three strains of Bifidobacteria and overexpression of luxS enhanced biofilm formation by Bifidobacterium longum NCC2705. In the present study we show that a functional luxS gene is widespread in the genus Bifidobacterium and that this gene in Bifidobacterium breve UCC2003 is involved in providing protection of Caenorhabditis elegans against Salmonella infection, a property which is linked to iron acquisition. Our data furthermore demonstrate that a functional luxS gene is required for murine gastrointestinal colonization by B. breve UCC2003. Members of the genus Bifidobacterium are recognized as being numerically dominant representatives of the microbiota of healthy breast-fed infants. Colonization of the newborn infant gut commences during and after birth by microbes from the mother and the environment.