The separation of sister chromatids in anaphase depends on the dissociation of cohesin from chromosomes. In vertebrates, some cohesin is removed from chromosomes at the onset of anaphase by proteolytic cleavage. In contrast, the bulk of cohesin is removed from chromosomes already in prophase and prometaphase by an unknown mechanism that does not involve cohesin cleavage. We show that Polo-like kinase is required for the cleavage-independent dissociation of cohesin from chromosomes in Xenopus. Cohesin phosphorylation depends on Polo-like kinase and reduces the ability of cohesin to bind to chromatin. These results suggest that Polo-like kinase regulates the dissociation of cohesin from chromosomes early in mitosis.