Galactooligosaccharides (GOS) are mixed oligosaccharides that are mainly composed of galactosyllactoses (GLs), which include 3′-GL, 4′-GL, and 6′-GL. Data from numerous in vitro and in vivo studies have shown that GOS selectively stimulate the growth of bifidobacteria. Previously, we identified the gene locus responsible for 4′-GL utilization, but the selective routes of uptake and catabolism of 3′- and 6′-GL remain to be elucidated. In this study, we used differential transcriptomics to identify the utilization pathways of these GLs within the Bifidobacterium breve YIT 4014T strain. We found that the BBBR_RS 2305–2320 gene locus, which includes a solute-binding protein (SBP) of an ATP-binding cassette (ABC) transporter and β-galactosidase, were up-regulated during 3′- and 6′-GL utilization. The substrate specificities of these proteins were further investigated, revealing that β-galactosidase hydrolyzed both 3′-GL and 6′-GL efficiently. Our surface plasmon resonance results indicated that the SBP bound strongly to 6′-GL, but bound less tightly to 3′-GL. Therefore, we looked for the other SBPs for 3′-GL and found that the BBBR_RS08090 SBP may participate in 3′-GL transportation. We also investigated the distribution of these genes in 17 bifidobacterial strains, including 9 B. breve strains, and found that the β-galactosidase genes were present in most bifidobacteria. Homologues of two ABC transporter SBP genes were found in all B. breve strains and in some bifidobacteria that are commonly present in the human gut microbiota. These results provide insights into the ability of human-resident bifidobacteria to utilize the main component of GOS in the gastrointestinal tract.