Xylo-oligosaccharides (XOS) are sugar oligomers of β-1,4-linked xylopyranosyl moieties which exert bifidogenic effect and are increasingly used as prebiotics. The kinetics and the metabolism of Bifidobacterium adolescentis DSMZ 18350 growing on XOS and xylose were investigated. The growth rate was higher on XOS, but greater biomass yield was attained on xylose. Unlike other prebiotics, XOS oligomers were utilized simultaneously, regardless of their chain length. Throughout XOS utilization, xylose concentration slightly increased, being not neatly consumed and remaining unfermented. During growth on XOS, β-xylosidase activity was present in the cytosol, but it occurred in the supernatant as well. A β-1,4-xylolytic enzyme was purified from the supernatant of XOS cultures. The enzyme, a homotetramer of a 39-kDa single protein, was capable of complete XOS hydrolysis and exhibited maximum activity at pH 6.0 and 55 °C. Based on the molecular weight, the protein can be ascribable to the product of the gene BAD_1527, the activity of which has been inferred as an endo-β-1,4-xylanase, but has not been characterized so far. This β-1,4-xylolytic enzyme, found to be active in the cultural supernatant, gives a reason for the never explained accumulation of the monosaccharides in the media of bifidobacterial cultures growing on XOS, without excluding the major role of the intracellular hydrolysis of the imported oligomers.