The DNA damage response (DDR) is activated upon DNA damage and prevents accumulation of mutations and chromosomal rearrangements, both driving carcinogenesis. Tumor cells often have defects in the DDR, which in combination with continuous cell proliferation are exploited by genotoxic cancer therapies. Most cancers, overcome initial sensitivity and develop drug resistance, e.g. by modulation of the DDR. Not much is known, however, about DNA damage responsive microRNAs in cancer therapy resistance. Therefore, we mapped temporal microRNA expression changes in primary breast epithelial cells upon low and high dose exposure to the DNA damaging agents ionizing radiation and cisplatin. A third of all DDR microRNAs commonly regulated across all treatments was also misexpressed in breast cancer, indicating a DDR defect. We repeated this approach in primary lung epithelial cells and non-small cell lung cancer samples and found that more than 40% of all DDR microRNAs was deregulated in non-small cell lung cancer. Strikingly, the microRNA response upon genotoxic stress in primary breast and lung epithelial cells was markedly different, although the biological outcome of DNA damage signaling (cell death/senescence or survival) was similar. Several DDR microRNAs deregulated in cancer modulated sensitivity to anti-cancer agents. In addition we were able to distinguish between microRNAs that induced resistance by potentially inducing quiescence (miR-296-5p and miR-382) or enhancing DNA repair or increased DNA damage tolerance (miR-21). In conclusion, we provide evidence that DNA damage responsive microRNAs are frequently misexpressed in human cancer and can modulate chemotherapy sensitivity.