Ku plays a key role in multiple nuclear processes, e.g., DNA repair, transcription regulation, and replication. It is believed that heterodimerization between Ku70 and Ku80 is essential for Ku-dependent DNA repair, although its role is poorly understood. We previously identified the Ku70-binding site of Ku80. In this study, to understand the role of heterodimerization in the function of Ku, we generated and/or analyzed cell lines stably expressing the EGFP-tagged-wild-type human Ku80, its Ku70-binding mutant, its NLS-dysfunctional mutant, or its double mutant in Ku80-deficient cells. Our results show that the Ku70-binding site of Ku80 is required for the stabilization of Ku70 in the cytoplasm and for the nuclear translocation of Ku80 through its heterodimerization with Ku70. In addition, our results suggest that the nuclear translocation of Ku80 through the Ku70-binding site as well as through the NLS of Ku80 play, at least in part, a role in Ku80-dependent DNA repair. Furthermore, our results suggest the possibility that Ku80 has a DNA DSB repair function independent of Ku70 in the nuclei, in addition to that dependent on Ku70.