In biochemistry, an acetylesterase (EC 3.1.1.6) is a class of enzyme which catalyzes the hydrolysis of acetic esters into an alcohol and acetic acid:

${\displaystyle {\ce {R-OC(O)CH3 + H2O}}\quad {\xrightarrow[{\text{acetylesterase}}]{}}\quad {\ce {R-OH + HO-C(O)CH3}}}$

This enzyme belongs to the family of hydrolases, specifically those acting on carboxylic ester bonds (esterases). The systematic name of this enzyme class is acetic-ester acetylhydrolase. Other names in common use include C-esterase (in animal tissues), acetic ester hydrolase, chloroesterase, p-nitrophenyl acetate esterase, and citrus acetylesterase.

As of late 2007, 3 structures have been solved for this class of enzymes, with PDB accession codes 1BS9, 1G66, and 2AXE.