BRCA1 plays an important role in the homologous recombination (HR)-mediated DNA double-strand break (DSB) repair, but the mechanism is not clear. Here we describe that BRCA1 forms a complex with CtIP and Mre11/Rad50/Nbs1 (MRN) in a cell cycle dependent manner. Significantly, the complex formation, especially the ionizing radiation (IR)-enhanced association of BRCA1 with MRN, requires cyclin-dependent kinase (CDK) activity. CtIP directly interacts with Nbs1. The in vivo association of BRCA1 with MRN is largely dependent on the association of CtIP with the BRCT domains at the C-terminus of BRCA1, while the N-terminus of BRCA1 also contributes to its association with MRN. CtIP, as well as the interaction of BRCA1 with CtIP and MRN, is critical for IR-induced ssDNA formation and cellular resistance to radiation. Consistently, CtIP itself is required for efficient HR-mediated DSB repair, like BRCA1 and MRN. These studies suggest that the complex formation of BRCA1/CtIP/MRN is important for facilitating DSB resection to generate ssDNA that is needed for HR-mediated DSB repair. Since CDK is important for establishing IR-enhanced interaction of MRN with BRCA1, we propose that the cell cycle-dependent complex formation of BRCA1, CtIP and MRN contributes to the activation of HR-mediated DSB repair in the S and G2 phases of the cell cycle.