Centromeres are chromosomal sites for kinetochore assembly, which supports the attachment of spindle microtubules and chromosome segregation. The centromere is specified by the incorporation of the histone H3 variant CENP-A into nucleosomes. CENP-A interacts with components of the constitutive centromere-associated network (CCAN), which forms the foundation for kinetochore assembly. Sathyan et al. now show that the first amino-terminal Gly residue of CENP-A is trimethylated and that this modification is important for interactions between CENP-A and CCAN in human cell lines. Chromosome segregation defects were observed in CENP-A-depleted cells that express CENP-A mutants that cannot be methylated, which most likely result from aberrant kinetochore assembly. Upon concomitant p53 depletion, the cells exhibited increased proliferative potential, both in vitro and when transplanted into mice, indicating that loss of CENP-A methylation may promote tumorigenic growth.