The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is rapidly phosphorylated at the Thr-2609 cluster and Ser-2056 upon ionizing radiation (IR). Furthermore, DNA-PKcs phosphorylation at both regions is critical for its role in DNA double strand break (DSB) repair as well as cellular resistance to radiation. IR-induced DNA-PKcs phosphorylation at Thr-2609 and Ser-2056, however, exhibits distinct kinetics indicating that they are differentially regulated. Although DNA-PKcs autophosphorylates itself at Ser-2056 after IR, we have reported here that ATM mediates DNA-PKcs phosphorylation at Thr-2609 as well as at the adjacent (S/T)Q motifs within the Thr-2609 cluster. In addition, our data suggest that DNA-PKcs- and ATM-mediated DNA-PKcs phosphorylations are cooperative and required for the full activation of DNA-PKcs and the subsequent DSB repair. Elimination of DNA-PKcs phosphorylation at both regions severely compromises radioresistance and DSB repair. Finally, our result provides a possible mechanism for the direct involvement of ATM in non-homologous end joining-mediated DSB repair.