Stable transmission of genetic information during cell division requires faithful mitotic spindle assembly and chromosome segregation. The Ran GTPase plays a key role in mitotic spindle assembly. However, how the generation of a chemical gradient of Ran-GTP at the spindle is coupled to mitotic post-translational modifications has never been characterized. Here, we solved the complex structure of Ran with the nucleotide release factor Mog1 and delineated a novel mitosis-specific acetylation-regulated Ran–Mog1 interaction during chromosome segregation. Our structure-guided functional analyses revealed that Mog1 competes with RCC1 for Ran binding in a GTP/GDP-dependent manner. Biochemical characterization demonstrated that Mog1-bound Ran prevents RCC1 binding and subsequent GTP loading. Surprisingly, Ran is a bona fide substrate of TIP60, and the acetylation of Lys134 by TIP60 liberates Mog1 from Ran binding during mitosis. Importantly, this acetylation-elicited switch of Ran binding to RCC1 promotes high level of Ran-GTP, which is essential for chromosome alignment. These results establish a previously uncharacterized regulatory mechanism in which TIP60 provides a homeostatic control of Ran-GTP level by tuning Ran effector binding for chromosome segregation in mitosis.