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. Display omitted •CLOCK acetylates metabolic enzymes to modulate their activity•CLOCK drives the cycle of acetylation and activation of metabolic enzymes•Interaction between CLOCK and metabolic enzymes is rhythmic•Arginine biogenesis and ureagenesis are rhythmically controlled by ASS1 acetylation Lin et al. reported that CLOCK acetylates ASS1 to inactivate its function in arginine biosynthesis. ASS1 acetylation by CLOCK exhibits rhythmicity, possibly caused by rhythmic interaction between CLOCK and ASS1, leading to the cycle of ASS1 activation. Two more enzymes, NDUFA9 and IMPDH2, were recognized as acetylation substrates of CLOCK.