Two chitin deacetylases, Cda1 and Cda2, from C. cinerea were expressed and characterized. Cda1 preferably deacetylates the non-reducing end residue of (GlcNAc)2, the internal or non-reducing end residue of (GlcNAc)3, and the non-reducing residue of (GlcNAc)6 after deacetylating the internal residues. In contrast, Cda2 preferably deacetylates the reducing end residue of (GlcNAc)2, the internal or reducing end residue of (GlcNAc)3, and the reducing residue of (GlcNAc)6 after deacetylating the internal residues. Furthermore, Cda1 prefers chitohexaose with higher degrees of acetylation for deacetylation, while Cda2 shows a weaker preference for chitohexaose with varying degrees of acetylation. The predicted Cda1 structure shows more hydrophobic aromatic amino acids on the surface near subsite +1 in the active site than on the surface near subsite -1, whereas the predicted Cda2 structure has more hydrophobic aromatic amino acids on the surface near subsite -1 than on the surface near subsite +1, which may be the molecular basis of the distinctive catalytic features between Cda1 and Cda2. Notably, Cda1 has a high transcription level in the non-elongating basal stipe region, whereas Cda2 has a high transcription level in the elongating apical stipe region, and the transcription level of the former is approximately five times that of the latter. Correspondingly, the molar ratio of GlcN/GlcNAc increased from 0.15 in the cell wall of the apical stipe region to 0.22 in the cell wall of the basal stipe region. Different modes of action of Cda1 and Cda2 may be related to their functions in the different stipe regions.