In addition to responding to environmental entrainment with diurnal variation， metabolism is also tightly controlled by cell-autonomous circadian clock. Extensive studies have revealed key roles of transcription in circadian control. Post-transcriptional regulation for the rhythmic gating of metabolic enzymes remains elusive. Here， we show that arginine biosynthesis and subsequent ureagenesis are collectively regulated by CLOCK (circadian locomotor output cycles kaput) in circadian rhythms. Facilitated by BMAL1 (brain and muscle Arnt-like protein)， CLOCK directly acetylates K165 and K176 of argininosuccinate synthase (ASS1) to inactivate ASS1， which catalyzes the rate-limiting step of arginine biosynthesis. ASS1 acetylation by CLOCK exhibits circadian oscillation in human cells and mouse liver， possibly caused by rhythmic interaction between CLOCK and ASS1， leading to the circadian regulation of ASS1 and ureagenesis. Furthermore， we also identified NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 (NDUFA9) and inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) as acetylation substrates of CLOCK. Taken together， CLOCK modulates metabolic rhythmicity by acting as a rhythmic acetyl-transferase for metabolic enzymes.