DNA double-strand breaks (DSBs) play an important role in promoting genomic instability and cell death. The precise repair of DSBs is essential for maintaining genome integrity during cancer progression, and inducing genomic instability or blocking DNA repair is an important mechanism through which chemo/radiotherapies exert killing effects on cancer cells. The two main pathways that facilitate the repair of DSBs in cancer cells are homologous recombination (HR) and non-homologous end-joining (NHEJ). Accumulating data suggest that the acetylation and deacetylation of DSB repair proteins regulate the initiation and progression of the cellular response to DNA DSBs, which may further affect the chemosensitivity or radiosensitivity of cancer cells. Here, we focus on the role of acetylation/deacetylation in the regulation of ataxia-telangiectasia mutated, Rad51, and 53BP1 in the HR pathway, as well as the relevant roles of PARP1 and Ku70 in NHEJ. Notably, several histone deacetylase (HDAC) inhibitors targeting HR or NHEJ have been demonstrated to enhance chemo/radiosensitivity in preclinical studies. This review highlights the essential role of acetylation/deacetylation in the regulation of DSB repair proteins, suggesting that HDAC inhibitors targeting the HR or NHEJ pathways that downregulate DNA DSB repair genes may be worthwhile cancer therapeutic agents.