The molecular details and impact of oligosaccharide uptake by distinct human gut microbiota (HGM) are currently not well understood. Non-digestible dietary galacto-and gluco-␣-(1,6)-oligosaccharides from legumes and starch, respectively, are preferentially fermented by mainly bifidobacteria and lactobacilli in the human gut. Here we show that the solute binding protein (BlG16BP) associated with an ATP binding cassette (ABC) transporter from the probiotic Bifidobacterium animalis subsp. lactis Bl-04 binds ␣-(1,6)-linked glucosides and galactosides of varying size, linkage, and monosaccharide composition with preference for the trisaccharides raffinose and panose. This preference is also reflected in the ␣-(1,6)-galactoside uptake profile of the bacterium. Structures of BlG16BP in complex with raffinose and panose revealed the basis for the remarkable ligand binding plasticity of BlG16BP, which recognizes the nonreducing ␣-(1,6)-diglycoside in its ligands. BlG16BP homologues occur predominantly in bifidobacteria and a few Firmicutes but lack in other HGMs. Among seven bifidobacterial taxa, only those possessing this transporter displayed growth on ␣-(1,6)-glycosides. Competition assays revealed that the dominant HGM commensal Bacteroides ovatus was out-competed by B. animalis subsp. lactis Bl-04 in mixed cultures growing on raffinose, the preferred ligand for the BlG16BP. By comparison, B. ovatus mono-cultures grew very efficiently on this trisaccharide. These findings suggest that the ABC-mediated uptake of raffinose provides an important competitive advantage, particularly against dominant Bacteroides that lack glycan-specific ABC-transporters. This novel insight highlights the role of glycan transport in defining the metabolic specialization of gut bacteria.The gastrointestinal tract hosts a highly diverse microbial community referred to as the human gut microbiota (HGM) 3 (1). This community, which is established shortly after birth, develops rapidly to form one of the most densely populated ecological niches in nature by the age of 2-3 years. Despite millions of years of co-evolution with mammalian hosts (2), only recently has the profound impact of the gut microbiota on various aspects of human health, including metabolic and immune-disorders, colon cancer, and brain function, as well as rate of aging been established (3-5). Evidence is rapidly accumulating that advantage can be taken of the gut microbiota in diagnosis, treatment, and prevention of diseases and disorders. To realize this potential it is important to discern the complex metabolic interactions among different microbiota taxa, which promote a healthy composition and prevent imbalance (dysbiosis) associated with disease (6). Diet is a key effector of the composition of the HGM (7, 8), and the metabolism of glycans has been highlighted as pivotal in maintaining a healthy bacterial community (9). Raffinose family oligosaccharides (RFOs), containing ␣-(1,6)-galactosides (10), are abundant in soybeans and other legumes and seeds (11, 12), but are no...