A GH67 α-glucuronidase gene derived from Bacillus halodurans C-125 was expressed in E. coli to obtain a recombinant enzyme ( Bh GlcA67). Using the purified enzyme, the enzymatic properties and substrate specificities of the enzyme were investigated. Bh GlcA67 showed maximum activity at pH 5.4 and 45 °C. When Bh GlcA67 was incubated with birchwood, oat spelts, and cotton seed xylan, the enzyme did not release any glucuronic acid or 4- O -methyl-glucuronic acid from these substrates. Bh GlcA67 acted only on 4- O -methyl-α-D-glucuronopyranosyl-(1→2)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranose (MeGlcA³Xyl₃), which has a glucuronic acid side chain with a 4- O -methyl group located at its non-reducing end, but did not on β-D-xylopyranosyl-(1→4)-[4- O -methyl-α-D-glucuronopyranosyl-(l→2)]-β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-xylop- yranose (MeGlcA³Xyl₄) and α-D-glucuronopyranosyl-(l→2)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranose (GlcA³Xyl₃). The environment for recognizing the 4- O -methyl group of glucuronic acid was observed in all the crystal structures of reported GH67 glucuronidases, and the amino acids for discriminating the 4- O -methyl group of glucuronic acid were widely conserved in the primary sequences of the GH67 family, suggesting that the 4- O -methyl group is critical for the activities of the GH67 family.