Arabinoxylan arabinofuranohydrolase-D3 (AXHd3) from Bifidobacterium adolescentis releases only C3-linked arabinose residues from double-substituted xylose residues. A genomic library of B. adolescentis DSM20083 was screened for the presence of the axhD3 gene. Two plasmids were identified containing part of the axhD3 gene. The nucleotide sequences were combined and three open reading frames (ORFs) were found. The first ORF showed high homology with xylanases belonging to family 8 of the glycoside hydrolases and this gene was designated xylA. The second ORF was the axhD3 gene belonging to glycoside hydrolase family 43. The third ( partial) ORF coded for a putative carboxylesterase. The axhD3 gene was cloned and expressed in Escherichia coli. Several substrates were employed in the biochemical characterization of recombinant AXHd3. The enzyme showed the highest activity toward wheat arabinoxylan oligosaccharides. In addition, beta-xylanase from Trichoderma sp. was able to degrade soluble wheat arabinoxylan polymer to a higher extent, after pretreatment with recombinant AXHd3. Arabinoxylan oligosaccharides incubated with a combination of recombinant AXHd3 and an alpha-L-arabinofuranosidase from Aspergillus niger did not result in a higher maximal release of arabinose than incubation with these enzymes separately.