Lysine acetylation is involved in various biological processes and is considered a key reversible post-translational modification in the regulation of gene expression, enzyme activity, and subcellular localization. This post-translational modification is therefore highly relevant in the context of circadian biology, but its characterization on the proteome-wide scale and its circadian clock dependence are still poorly described. Here, we provide a comprehensive and rhythmic acetylome map of the mouse liver. Rhythmic acetylated proteins showed subcellular localization-specific phases that correlated with the related metabolites in the regulated pathways. Mitochondrial proteins were over-represented among the rhythmically acetylated proteins and were highly correlated with SIRT3-dependent deacetylation. SIRT3 activity being nicotinamide adenine dinucleotide (NAD) ⁺ level-dependent, we show that NAD ⁺ is orchestrated by both feeding rhythms and the circadian clock through the NAD ⁺ salvage pathway but also via the nicotinamide riboside pathway. Hence, the diurnal acetylome relies on a functional circadian clock and affects important diurnal metabolic pathways in the mouse liver.