Akt kinase-interacting protein 1 (Aki1)/Freud-1/CC2D1A is localized in the cytosol, nucleus, and centrosome. Aki1 plays distinct roles depending on its localization. In the cytosol, it acts as a scaffold protein in the phosphoinositide 3-kinase (PI3K)/3-phosphoinositide-dependent protein kinase 1 (PDK1)/Akt pathway. In the nucleus, it is a transcriptional repressor of the serotonin-1A (5-HT1A) receptor. In the centrosome, it regulates spindle pole localization of the cohesin subunit Scc1, thereby mediating centriole cohesion during mitosis. Although the function of Aki1 has been well clarified, the regulatory machinery of Aki1 is poorly understood. We previously found that Aki1 in mitotic cells displayed reduced mobility on immunoblot analysis, but the reason for this was unclear. Here we show that the electrophoretic mobility shift of Aki1 is derived from mitotic phosphorylation. The cyclin B1-cyclin-dependent kinase 1 (Cdk1) complex was found to be one of the kinases responsible for Aki1 phosphorylation during mitosis. We identified the Ser(208) residue of Aki1 as a cyclin B1-Cdk1 phosphorylation site. Furthermore, cyclin B1-Cdk1 inhibitor treatment was shown to attenuate the level of Aki1 in complex with Scc1, suggesting that Aki1 phosphorylation by cyclin B1-Cdk1 contributes to Aki1-Scc1 complex formation. Our results indicate that cyclin B1-Cdk1 is a kinase of Aki1 during mitosis and that its phosphorylation of Aki1 may regulate mitotic function.