In enzymology, a N-acetylglucosamine deacetylase (EC 3.5.1.33) is an enzyme that catalyzes the chemical reaction

N-acetyl-D-glucosamine + H₂O ${\displaystyle \rightleftharpoons }$ D-glucosamine + acetate

Thus, the two substrates of this enzyme are N-acetyl-D-glucosamine and H₂O, whereas its two products are D-glucosamine and acetate.

This enzyme belongs to the family of hydrolases, those acting on carbon-nitrogen bonds other than peptide bonds, specifically in linear amides. The systematic name of this enzyme class is N-acetyl-D-glucosamine amidohydrolase. Other names in common use include acetylaminodeoxyglucose acetylhydrolase, and N-acetyl-D-glucosaminyl N-deacetylase. This enzyme participates in aminosugars metabolism.

As of late 2007, two structures have been solved for this class of enzymes, with PDB accession codes 2C1G and 2C1I.

• Roseman S (May 1957). "Glucosamine metabolism. I. N-acetylglucosamine deacetylase". The Journal of Biological Chemistry. 226 (1): 115–24. doi:10.1016/S0021-9258(18)64810-0. PMID 13428742.