Post-translational phosphorylation plays critical roles in the assembly of signaling and repair proteins in the DNA damage response pathway. RAP80, a component of the BRCA1-A complex, is crucial in cell cycle checkpoint activation and DNA damage repair. However, its molecular mechanism is unclear. In this study, we identified Cdk1 as a new RAP80-binding protein and demonstrated that the Cdk1-cyclin B(1) complex phosphorylates RAP80 at Ser-677 using an in vitro kinase assay and a phosphopeptide-specific antibody against phospho-Ser-677 of RAP80. RAP80 Ser-677 phosphorylation occurred in the M phase of the cell cycle when Cdk1 was in an active state. In addition, ionizing radiation (IR) induced RAP80 phosphorylation at Ser-677. Mutation of Ser-677 to alanine sensitized cells to IR and functioned in G(2)/M checkpoint control. These results suggest that post-translational phosphorylation of RAP80 by the Cdk1-cyclin B(1) complex is important for RAP80 functional sensitivity to IR and G(2)/M checkpoint control.