Faithful chromosome segregation during mitosis is critical for maintaining genome integrity in cell progeny and relies on accurate and robust kinetochore-microtubule attachments. The NDC80 complex, a tetramer comprising kinetochore protein HEC1 (HEC1), NDC80 kinetochore complex component NUF2 (NUF2), NDC80 kinetochore complex component SPC24 (SPC24), and SPC25, plays a critical role in kinetochore-microtubule attachment. Mounting evidence indicates that phosphorylation of HEC1 is important for regulating the binding of the NDC80 complex to microtubules. However, it remains unclear whether other post-translational modifications, such as acetylation, regulate NDC80-microtubule attachment during mitosis. Here, using pulldown assays with Hela cell lysates and site-directed mutagenesis, we show that HEC1 is a bona fide substrate of the lysine acetyltransferase Tat-interacting protein, 60KD(TIP60) and that TIP60-mediated acetylation of HEC1 is essential for accurate chromosome segregation in mitosis. We demonstrate that TIP60 regulates the dynamic interactions between NDC80 and spindle microtubules during mitosis and observed that TIP60 acetylates HEC1 at two evolutionarily conserved residues Lys-53 and Lys-59. Importantly, this acetylation weakened the phosphorylation of the N-terminal HEC11-80 region at Ser-55 and Ser-62, which is governed by Aurora B and regulates NDC80-microtubule dynamics, indicating functional cross-talk between these two post-translational modifications of HEC1. Moreover, the TIP60-mediated acetylation was specifically reversed by sirtuin1 (SIRT1). Taken together, our results define a conserved signaling hierarchy, involving HEC1, TIP60, Aurora B, and SIRT1, which integrates dynamic HEC1 acetylation and phosphorylation for accurate kinetochore- microtubule attachment in the maintenance of genomic stability during mitosis.