Evolutionarily conserved polo-like kinase, Cdc5 (Plk1 in humans), associates with kinetochores during mitosis; however, the role of cell cycle–dependent centromeric ( CEN) association of Cdc5 and its substrates that exclusively localize to the kinetochore have not been characterized. Here we report that evolutionarily conserved CEN histone H3 variant, Cse4 (CENP-A in humans), is a substrate of Cdc5, and that the cell cycle–regulated association of Cse4 with Cdc5 is required for cell growth. Cdc5 contributes to Cse4 phosphorylation in vivo and interacts with Cse4 in mitotic cells. Mass spectrometry analysis of in vitro kinase assays showed that Cdc5 phosphorylates nine serine residues clustered within the N-terminus of Cse4. Strains with cse4-9SA exhibit increased errors in chromosome segregation, reduced levels of CEN-associated Mif2 and Mcd1/Scc1 when combined with a deletion of MCM21. Moreover, the loss of Cdc5 from the CEN chromatin contributes to defects in kinetochore integrity and reduction in CEN-associated Cse4. The cell cycle–regulated association of Cdc5 with Cse4 is essential for cell viability as constitutive association of Cdc5 with Cse4 at the kinetochore leads to growth defects. In summary, our results have defined a role for Cdc5-mediated Cse4 phosphorylation in faithful chromosome segregation.