Shortened kinetochore microtubules take separated chromatids to the opposing spindle poles in anaphase. Fission yeast Dis1 belongs to the Dis1/XMAP215/TOG family that is required for proper microtubule dynamics. Here, we report that Dis1is regulated by Cdc2 phosphorylation and that this mitotic phosphorylation ensures the fidelity of chromosome segregation. Whereas mutants Dis1(6A) and Dis1(6E) that substitute all of the six Cdc2 sites for Ala or Glu, respectively, produce colonies at 22 degrees C-36 degrees C, Dis1(6A) but not Dis1(6E) loses a minichromosome and reveals aberrant chromosome segregation at significant frequencies. Dis1(WT) is recruited to two regions of the mitotic spindle: kinetochores (possibly also kinetochore microtubules) in metaphase and the pole-to-pole microtubule lattice in anaphase. Mutant Dis1(6E) preferentially binds to metaphase kinetochores, whereas Dis1(6A), which is located along microtubules, fails in its accumulation at kinetochores. Dis1(6A) displays synthetic lethality with the mis12-537, which is a mutant that compromises kinetochore function. Dis1(6E) mimics the Cdc2-phosphorylated form of Dis1(WT), whereas Dis1(6A) can partially rescue the phenotype resulting form deletion of Mtc1/Alp14, another XMAP215-like protein. In anaphase, dephosphorylated Dis1 and Dis1(6A), but not Dis1(6E), move to the spindle microtubule lattice near the SPBs. Cdc2 thus directly phosphorylates Dis1, and this phosphorylation regulates Dis1 localization in both metaphase and anaphase and ensures high-fidelity segregation.