Acetylation (or in IUPAC nomenclature ethanoylation) describes a reaction that introduces an acetyl functional group into a chemical compound. (Deacetylation is the removal of the acetyl group.)

Specifically, acetylation refers to that process of introducing an acetyl group (resulting in an acetoxy group) into a compound, to be specific, the substitution of an acetyl group for an active hydrogen atom. A reaction involving the replacement of the hydrogen atom of a hydroxyl group with an acetyl group (CH₃ CO) yields a specific ester, the acetate. Acetic anhydride is commonly used as an acetylating agent reacting with free hydroxyl groups. For example, it is used in the synthesis of aspirin and heroin.

In biology, i.e., in living cells, acetylation occurs as a co-translational and post-translational modification of proteins, for example, histones, p53, and tubulins. In fact, proteomics studies have identified thousands of acetylated mammalian proteins . Among these proteins, chromatin proteins and metabolic enzymes are highly represented, indicating that acetylation has a considerable impact on gene expression and metabolism. In bacteria, 90% of proteins involved in central metabolism of Salmonella enterica are acetylated .