Xylooligosaccharides have strong bifidogenic properties and are increasingly used as a prebiotic. Nonetheless, little is known about the degradation of these substrates by bifidobacteria. We characterized two recombinant β-xylosidases, XylB and XylC, with different substrate specificities from Bifidobacterium adolescentis. XylB is a novel β-xylosidase that belongs to the recently introduced glycoside hydrolase family 120. In contrast to most reported β-xylosidases, it shows only weak activity on xylobiose and prefers xylooligosaccharides with a degree of polymerization above two. The remaining xylobiose is efficiently hydrolyzed by the second B. adolescentis β-xylosidase, XylC, a glycoside hydrolase of family 43. Furthermore, XylB releases more xylose from arabinose-substituted xylooligosaccharides than XylC (30% and 20%, respectively). The different specificities of XylB, XylC, and the recently described reducing-end xylose-releasing exo-oligoxylanase RexA show how B. adolescentis can efficiently degrade prebiotic xylooligosaccharides.