To ensure genome integrity, DNA replication takes place only once per cell cycle and is tightly controlled by cyclin-dependent kinase (Cdk1). Cdc6p is part of the prereplicative complex, which is essential for DNA replication. Cdc6 is phosphorylated by cyclin-Cdk1 to promote its degradation after origin firing to prevent DNA rereplication. We previously showed that a yeast GSK-3 homologue, Mck1 kinase, promotes Cdc6 degradation in a SCF(Cdc4)-dependent manner, therefore preventing rereplication. Here we present evidence that Mck1 directly phosphorylates a GSK-3 consensus site in the C-terminus of Cdc6. The Mck1-dependent Cdc6 phosphorylation required priming by cyclin/Cdk1 at an adjacent CDK consensus site. The sequential phosphorylation by Mck1 and Clb2/Cdk1 generated a Cdc4 E3 ubiquitin ligase-binding motif to promote Cdc6 degradation during mitosis. We further revealed that Cdc6 degradation triggered by Mck1 kinase was enhanced upon DNA damage caused by the alkylating agent methyl methanesulfonate and that the resulting degradation was mediated through Cdc4. Thus, Mck1 kinase ensures proper DNA replication, prevents DNA damage, and maintains genome integrity by inhibiting Cdc6.