Although many xylanases have been studied, many of the characteristics of xylanases toward branches in xylan remain unclear. In this study, the substrate specificity of a GH11 xylanase from Streptomyces olivaceoviridis E-86 (SoXyn11B) was elucidated based on its three-dimensional structure. Subsite mapping suggests that SoXyn11B has seven subsites (four subsites on the - side and three subsites on the + side), and it is one longer than the GH10 xylanase from S. olivaceoviridis (SoXyn10A). SoXyn11B has no affinity for the subsites at either end of the scissile glycosidic bond, and the sugar-binding energy at subsite - 2 was the highest, followed by subsite + 2. These properties were very similar to those of SoXyn10A. In contrast, SoXyn11B produced different branched oligosaccharides from bagasse compared with those of SoXyn10A. These branched oligosaccharides were identified as O-beta-D-xylopyranosyl-(1 -> 4)-[O-alpha-L-arabinofuranosyl-(1 -> 3)]-O-beta-D-xylopyranosyl-(1 -> 4)-beta-D-xylopyranosyl-(1 -> 4)-beta-D-xylopyranose (Ara(3)Xyl(4)) and O-beta-D-xylopyranosyl-(1 -> 4)-[O-4-O-methyl-alpha-D-glucuronopyranosyl-(l -> 2)]-beta-D-xylopyranosyl-(1 -> 4)-beta-D-xylopyranosyl-(1 -> 4)-beta-D-xylopyranose (MeGlcA(3)Xyl(4)) by nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS) and confirmed by crystal structure analysis of SoXyn11B in complex with these branched xylooligosaccharides. SoXyn11B has a beta-jerryroll fold structure, and the catalytic cleft is located on the inner beta-sheet of the fold. The ligand-binding structures revealed seven subsites of SoXyn11B. The 2- and 3-hydroxy groups of xylose at the subsites + 3, + 2, and - 3 face outwards, and an arabinose or a glucuronic acid side chain can be linked to these positions. These subsite structures appear to cause the limited substrate specificity of SoXyn11B for branched xylooligosaccharides. Key points Crystal structure of family 11 beta-xylanase from Streptomyces olivaceoviridis was determined. Topology of substrate-binding cleft of family 11 beta-xylanase from Streptomyces olivaceoviridis was characterized. Mode of action of family 11 beta-xylanase from Streptomyces olivaceoviridis for substitutions in xylan was elucidated.