DNA topoisomerase II (topo II) regulates the topological state of DNA and is necessary for DNA replication, transcription, and chromosome segregation. Topo II has essential functions in cell proliferation and therefore is a critical target of anticancer drugs. In this study, using Phos-tag SDS-PAGE analysis in fission yeast ( Schizosaccharomyces pombe ), we identified casein kinase II (Cka1/CKII)-dependent phosphorylation at the C-terminal residues Ser 1363  and Ser 1364  in topo II. We found that this phosphorylation decreases the inhibitory effect of an anticancer catalytic inhibitor of topo II, ICRF-193, on mitosis. Consistent with the constitutive activity of Cka1/CKII, Ser 1363  and Ser 1364  phosphorylation of topo II was stably maintained throughout the cell cycle. We demonstrate that ICRF-193-induced chromosomal mis-segregation is further exacerbated in two temperature-sensitive mutants,  cka1-372  and  cka1/orb5-19 , of the catalytic subunit of CKII or in the topo II nonphosphorylatable alanine double mutant  top2-S1363A,S1364A  but not in cells of the phosphomimetic glutamate double mutant  top2-S1363E,S1364E  Our results suggest that Ser 1363  and Ser 1364  in topo II are targeted by Cka1/CKII kinase and that their phosphorylation facilitates topo II ATPase activity in the N-terminal region, which regulates protein turnover on chromosome DNA. Because CKII-mediated phosphorylation of the topo II C-terminal domain appears to be evolutionarily conserved, including in humans, we propose that attenuation of CKII-controlled topo II phosphorylation along with catalytic topo II inhibition may promote anticancer effects.