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Acetylated glucuronoxylan is one of the most common types of hemicellulose in nature. The structure is formed by a β-(1→4)-linked <span style="font-variant: small-caps;">D-</span>xylopyranosyl (Xyl<i>p</i>) backbone that can be substituted with an acetyl group at <i>O</i>-2 and <i>O-</i>3 positions, and α-(1→2)-linked 4-<i>O</i>-methylglucopyranosyluronic acid (MeGlc<i>p</i>A). Acetyl xylan esterases (AcXE) that target mono- or doubly acetylated Xyl<i>p</i> are well characterized; however, the previously studied AcXE from <i>Flavobacterium johnsoniae</i> (<i>Fjo</i>AcXE) was the first to remove the acetyl group from 2-<i>O</i>-MeGlc<i>p</i>A-3-<i>O</i>-acetyl-substituted Xyl<i>p</i> units, yet structural characteristics of these enzymes remain unspecified. Here, six homologs of <i>Fjo</i>AcXE were produced and three crystal structures of the enzymes were solved. Two of them are complex structures, one with bound MeGlc<i>p</i>A and another with acetate. All homologs were confirmed to release acetate from 2-<i>O</i>-MeGlc<i>p</i>A-3-<i>O</i>-acetyl-substituted xylan, and the crystal structures point to key structural elements that might serve as defining features of this unclassified carbohydrate esterase family. Enzymes comprised two domains: N-terminal CBM domain and a C-terminal SGNH domain. In <i>Fjo</i>AcXE and all studied homologs, the sequence motif around the catalytic serine is Gly-Asn-Ser-Ile (GNSI), which differs from other SGNH hydrolases. Binding by the MeGlc<i>p</i>A-Xyl<i>p</i> ligand is directed by positively charged and highly conserved residues at the interface of the CBM and SGNH domains of the enzyme.