AbstractIn plant cell walls, the hydroxyproline‐rich glycoproteins (HRGPs) such as extensin contain oligoarabinofuranoside linked to a hydroxyproline (Hyp) residue. The mature arabinooligosaccharide was revealed to be a tetrasaccharide (α‐l‐Araf‐(1→3)‐β‐l‐Araf‐(1→2)‐β‐l‐Araf‐(1→2)‐β‐l‐Araf, l‐Araf₄), whose linkages are targets of the bifidobacterial and Xanthomonas arabinooligosaccharide‐degrading enzymes. The l‐Araf₄ motif was cleaved by GH43 α‐l‐arabinofuranosidase (Arafase) and converted to an l‐Araf₃‐linked structure. The latter is then cleaved by GH121 β‐l‐arabinobiosidase (HypBA2), producing β‐l‐Araf‐(1→2)‐l‐Ara (β‐l‐arabinobiose) and mono‐β‐l‐Araf linked to the HRGP backbone. In bifidobacteria, the β‐l‐arabinobiose is then hydrolyzed by GH127 β‐l‐Arafase (Bll1HypBA1), a mechanistically unique cysteine glycosidase. We recently identified the distantly related homologue from Xanthomonas euvesicatoria as GH146 β‐l‐Arafase along with paralogues from Bifidobacterium longum, one of which, Bll4HypBA1 (BLLJ_0089), can degrade l‐Araf₁‐Hyp in a similar way to that of GH146. As the chemical synthesis of the extensin hydrophilic motif 1 a, which possesses three distinct linkages that connect four oligoAraf residues [Hyp(l‐Araf_n) (n=4, 3, 1)], was achieved previously, we precisely monitored the step‐wise enzymatic cleavage of 1 a in addition to that of potato lectin. The results unequivocally revealed that this enzyme specifically degrades the Hyp(l‐Araf₁) motif.