ARTICLE ABSTRACTProgression of mitotic cell cycle and chromosome condensation and segregation are controlled by posttranslational protein modifications such as phosphorylation and SUMOylation. However, how SUMO isopeptidases (SENP) regulate cell mitotic procession is largely unknown. Here, we demonstrate that precise phosphorylation of SENP3 during mitosis suppresses SENP3 deSUMOylation activity towards chromosome-associated proteins, including topoisomerase IIα (TopoIIα). Cyclin B-dependent kinases 1 and protein phosphatase 1α were identified as the kinase and phosphatase in control of mitotic SENP3 phosphorylation, respectively. SENP3 phosphorylation decreased its interaction with TopoIIα, resulting in reduced SENP3 deSUMOylation activity on TopoIIα. Furthermore, we observed mitotic arrest, increased chromosome instability, and promotion of tumorigenesis in cells expressing a nonphosphorylatable SENP3 mutant. These data show that SENP3 phosphorylation plays a crucial role in regulating the SUMOylation of chromosome-associated proteins and chromosome stability in mitosis.Significance: Phosphorylation of SENP3 regulates SUMOylation of chromosome-associated proteins to maintain genomic stability during mitosis. Cancer Res; 78(9); 2171–8. ©2018 AACR.