The stem cell genomic stability forms the basis for robust tissue homeostasis, particularly in high-turnover tissues. For the genomic stability, DNA damage response (DDR) is essential. This study was focused on role of the MRN complex (Mre11, Rad50, and Nbs1), two major DDR-related factors, ataxia telangiectasia-mutated (ATM) and ATM- and RAD3-related (ATR) kinases, and two transducer, Chk1 and Chk2 in the maintenance of intestinal stem cells in the adult Drosophila midgut. We explored the role of DNA damage response-related factors, utilizing immunostaining with an anti-pS/TQ antibody as an indicator of ATM/ATR activation, γ-irradiation as a DNA damage inducer, and the UAS/GAL4 system for cell type-specific knockdown of DNA damage response-related factors, or both during adulthood. Here we show that DDR is activated in the intestinal stem cells and enterocytes by DNA damage. ISCs or ECs-specific knockdown of DDR factors caused ISC or EC cell death. and induced intestinal stem cell proliferation. The results showed that the pS/TQ signals got stronger with age and after oxidative stress. The pS/TQ signals were found to be more dependent on ATR rather than on ATM in ISCs/enteroblasts. Furthermore, an ISC/EB-specific knockdown of DNA damage response-related factors decreased the number of ISCs and oxidative stress-induced ISC proliferation. EC-specific knockdown of DNA damage response-related factors increased ISC proliferation and centrosome amplification. These results indicate that DNA damage response-related factor is essential for the maintenance of intestinal homeostasis with age.