Precise chromosome segregation is mediated by a well-assembled mitotic spindle, which requires balance of the kinase activity of Aurora A (AurA, also known as AURKA). However, how this kinase activity is regulated remains largely unclear. Here, using in vivo and in vitro assays, we report that conjugation of SUMO2 with AurA at K258 in early mitosis promotes the kinase activity of AurA and facilitates the binding with its activator Bora. Knockdown of the SUMO proteases SENP3 and SENP5 disrupts the deSUMOylation of AurA, leading to increased kinase activity and abnormalities in spindle assembly and chromosome segregation, which could be rescued by suppressing the kinase activity of AurA. Collectively, these results demonstrate that SENP3 and SENP5 deSUMOylate AurA to render spatiotemporal control on its kinase activity in mitosis.