Ornithine carbamoyltransferase (OTC) is a key enzyme in the urea cycle to detoxify ammonium produced from amino acid catabolism. OTC deficiency is an X-linked genetic disorder ranging from fatal in newborns to hyperammonemia and anorexia in adults. Through affinity purification of acetylated peptides and mass spectrometry, we identified that OTC is acetylated on lysine residues, including Lys88, which is also mutated in OTC-deficient patients. OTC acetylation was confirmed to occur under physiological conditions. Biochemical characterizations revealed that OTC Lys88 acetylation decreases the affinity for carbamoyl phosphate, one of the two OTC substrates, and the maximum velocity, whereas the K(m) for ornithine, the other OTC substrate, is not affected. Furthermore, Lys88 acetylation is regulated by both extracellular glucose and amino acid availability, indicating that OTC activity may be regulated by cellular metabolic status. Our results provide an example of the novel mechanism of regulating metabolic enzyme activity through protein acetylation.