The β-glucans are structurally varied, naturally occurring components of the cell walls, and storage materials of a variety of plant and microbial species. In the human diet, mixed-linkage glucans [MLG - β-(1,3/4)-glucans] influence the gut microbiome and the host immune system. Although consumed daily, the molecular mechanism by which human gut Gram-positive bacteria utilize MLG largely remains unknown. In this study, we usedBlautia productaATCC 27340 as a model organism to develop an understanding of MLG utilization.B. productaencodes a gene locus comprising a multi-modular cell-anchored endo-glucanase (BpGH16MLG), an ABC transporter, and a glycoside phosphorylase (BpGH94MLG) for utilizing MLG, as evidenced by the upregulation of expression of the enzyme- and solute binding protein (SBP)–encoding genes in this cluster when the organism is grown on MLG. We determined that recombinantBpGH16MLGcleaved various types of β-glucan, generating oligosaccharides suitable for cellular uptake byB. producta. Cytoplasmic digestion of these oligosaccharides is then performed by recombinantBpGH94MLGand β-glucosidases (BpGH3-AR8MLGandBpGH3-X62MLG). Using targeted deletion, we demonstratedBpSBPMLGis essential forB. productagrowth on barley β-glucan. Furthermore, we revealed that beneficial bacteria, such asRoseburia faecisJCM 17581T,Bifidobacterium pseudocatenulatumJCM 1200T,Bifidobacterium adolescentisJCM 1275T, andBifidobacterium bifidumJCM 1254, can also utilize oligosaccharides resulting from the action ofBpGH16MLG. Disentangling the β-glucan utilizing the capability ofB. productaprovides a rational basis on which to consider the probiotic potential of this class of organism.