Phosphofructokinase (Pfk) catalyzes the phosphorylation of fructose 6‐phosphate and is a key regulatory point in the glycolysis pathway. Multiple lysine residues in both Pfk isozymes, PfkA and PfkB, have been identified to be acetylated in Escherichia coli by proteomic studies, but no studies have been implemented to further characterize these acetylation events. To investigate the role of Pfk acetylation, the genetic code expansion strategy was used to generate homogeneously acetylated Pfk variants at target lysine sites that have been reported to be acetylated in nature. We found that acetylation of K309 of PfkA and K27 of PfkB decreased PfK enzyme activities significantly. We further investigated the deacetylation and acetylation processes of Pfk isozymes biochemically and genetically. Acetyl phosphate‐mediated non‐enzymatic acetylation could be the major mechanism of Pfk isozyme acetylation in E. coli , whereas NAD‐dependent protein deacylase CobB can remove most of the acetylated lysine residues but not K309 of PfkA and K27 of PfkB, which affect enzyme activities. Because of the important role of Pfk in cellular metabolism, the results of the present study are expected to facilitate studies in the fields of metabolic engineering and research.