ATM is a member of phosphatidylinositol 3-kinase-related kinase (PIKK) family and plays a critical role in DNA double-strand break repair. TRRAP, a unique kinase-less member of PIKK family associates with p400 ATPase and TIP60 histone acetyltransferase and participates in DNA damage response. Here we investigate a possibility as to whether ATM directly interacts with p400 for an efficient DNA damage response. Our data indicate that ATM and p400 associate constitutively regardless of DNA damage state and that the N-terminal domain of p400 is vital for this interaction. Heterologous expression studies using Sf9 cells revealed that the complex can be reconstituted without other mammalian bridging proteins. Consistent with a direct association, the presence of cytoplasmic p400 in insect cells is sufficient to progressively deplete nuclear ATM, sequestering it outside of the nucleus. Over-expression of ATM-interacting p400 regions in U2OS cells induced dominant negative effects including the inhibition of both DNA damage repair and cell proliferation. Consistent with the dominant negative effect, the stable expression of an N-terminal p400 fragment showed a decrease in the association of p400 with ATM, but did not alter the association of p400 with TRRAP. Taken together, our findings suggest that a protein-protein interaction between ATM and p400 ATPase occurs independently of DNA damage and contributes to efficient DNA damage response and repair.