Non-homologous end joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs) generated by ionizing radiation (IR) and anti-cancer drugs. Therefore, inhibiting the activity of proteins involved in this pathway is a promising way of sensitizing cancer cells to both radiotherapy and chemotherapy. In this study, we developed an assay for evaluating NHEJ activity against DSBs in chromosomal DNA in human cells to identify the chromatin modification/remodeling proteins involved in NHEJ. We showed that ablating the activity of the homologous histone acetyltransferases, CBP and p300, using inhibitors or small interfering RNAs-suppressed NHEJ. Ablation of CBP or p300 impaired IR-induced DSB repair and sensitized lung cancer cells to IR and the anti-cancer drug, etoposide, which induces DSBs that are repaired by NHEJ. The CBP/p300 proteins were recruited to sites of DSBs and their ablation suppressed acetylation of lysine 18 within histone H3, and lysines 5, 8, 12, and 16 within histone H4, at the DSB sites. This then suppressed the recruitment of KU70 and KU80, both key proteins for NHEJ, to the DSB sites. Ablation of CBP/p300 also impaired the recruitment of BRM, a catalytic subunit of the SWI/SNF complex involved in chromatin remodeling at DSB sites. These results indicate that CBP and p300 function as histone H3 and H4 acetyltransferases at DSB sites in NHEJ and facilitate chromatin relaxation. Therefore, inhibition CBP and p300 activity may sensitize cancer cells to radiotherapy and chemotherapy.