O-linked　N-acetyl-glucosamine　glycosylation　(O-GlcNAcylation)　of　intracellular　proteins　is　a　dynamic　process　broadly　implicated　in　age-related　disease,　yet　it　remains　uncharacterized　whether　and　how　O-GlcNAcylation　contributes　to　the　natural　aging　process.　O-GlcNAc　transferase　(OGT)　and　the　opposing　enzyme　O-GlcNAcase　(OGA)　control　this　nutrient-sensing　protein　modification　in　cells.　Here,　we　show　that　global　O-GlcNAc　levels　are　increased　in　multiple　tissues　of　aged　mice.　In　aged　liver,　carbamoyl　phosphate　synthetase　1　(CPS1)　is　among　the　most　heavily　O-GlcNAcylated　proteins.　CPS1　O-GlcNAcylation　is　reversed　by　calorie　restriction　and　is　sensitive　to　genetic　and　pharmacological　manipulations　of　the　O-GlcNAc　pathway.　High　glucose　stimulates　CPS1　O-GlcNAcylation　and　inhibits　CPS1　activity.　Liver-specific　deletion　of　OGT　potentiates　CPS1　activity　and　renders　CPS1　irresponsive　to　further　stimulation　by　a　prolonged　fasting.　Our　results　identify　CPS1　O-GlcNAcylation　as　a　key　nutrient-sensing　regulatory　step　in　the　urea　cycle　during　aging　and　dietary　restriction,　implying　a　role　for　mitochondrial　O-GlcNAcylation　in　nutritional　regulation　of　longevity.