Double strand break (DSB) recognition is the first step in the DSB damage response and involves activation of ataxia telangiectasia-mutated (ATM) and phosphorylation of targets such as p53 to trigger cell cycle arrest, DNA repair, or apoptosis. It was reported that activation of ATM- and Rad3-related (ATR) kinase by DSBs also occurs in an ATM-dependent manner. On the other hand, Ku70/80 is known to participate at a later time point in the DSB response, recruiting DNA-PKcs to facilitate non-homologous end joining. Because Ku70/80 has a high affinity for broken DNA ends and is abundant in nuclei, we examined their possible involvement in other aspects of the DSB damage response, particularly in modulating the activity of ATM and other phosphatidylinositol (PI) 3-related kinases during DSB recognition. We thus analyzed p53(Ser18) phosphorylation in irradiated Ku-deficient cells and observed persistent phosphorylation in these cells relative to wild type cells. ATM or ATR inhibition revealed that this phosphorylation is mainly mediated by ATM-dependent ATR activity at 2 h post-ionizing radiation in wild type cells, whereas in Ku-deficient cells, this occurs mainly through direct ATM activity, with a secondary contribution from ATR via a novel ATM-independent mechanism. Using ATM/Ku70 double-null cell lines, which we generated, we confirmed that ATM-independent ATR activity contributed to persistent phosphorylation of p53(Ser18) in Ku-deficient cells at 12 h post-ionizing radiation. In summary, we discovered a novel role for Ku70/80 in modulating ATM-dependent ATR activation during DSB damage response and demonstrated that these proteins confer a protective effect against ATM-independent ATR activation at later stages of the DSB damage response.