We characterized a β-L-arabinopyranosidase AbpBL (BLLJ_1823) belonging to the glycoside hydrolase family 27 (GH27) from Bifidobacterium longum subsp. longum JCM1217. The recombinant AbpBL expressed in Escherichia coli hydrolyzed pNP-β-L-arabinopyranoside but not pNP-α-D-galactopyranoside. The enzyme also liberated L-arabinose from the β-L-arabinopyranosyl side chain of larch wood arabinogalactan. However, we could not detect any β-L-arabinopyranosidase activity or remarkable transcriptional induction in cultured cells of B. longum subsp. longum. Mutagenesis experiments revealed that I56D and I56A mutants both exhibited β-L-arabinopyranosidase and α-D-galactopyranosidase activities. AbpBL Ile-56 residue is a critical residue for the specificity of β-L-arabinopyranosidase.