In eukaryotic cells, the cell-division cycle (CDC)-6 protein is essential to promote the assembly of pre-replicative complexes in the early G1 phase of the cell cycle, a process requiring tight regulation to ensure that proper origin licensing occurs once per cell cycle. Here we show that, in late G1 and early S phase, CDC6 is found in a complex also containing Cyclin A, cyclin-dependent kinase (CDK)-2 and the acetyltransferase general control nonderepressible 5 (GCN5). GCN5 specifically acetylates CDC6 at three lysine residues flanking its cyclin-docking motif, and this modification is crucial for the subsequent phosphorylation of the protein by Cyclin A–CDKs at a specific residue close to the acetylation site. GCN5-mediated acetylation and site-specific phosphorylation of CDC6 are both necessary for the relocalization of the protein to the cell cytoplasm in the S phase, as well as to regulate its stability. This two-step, intramolecular regulatory program by sequential modification of CDC6 seems to be essential for proper S-phase progression.