This study reports a histone deacetylation-independent mechanism whereby histone deacetylase (HDAC) inhibitors sensitize prostate cancer cells to DNA-damaging agents by targeting Ku70 acetylation. Ku70 represents a crucial component of the nonhomologous end joining repair machinery for DNA double-strand breaks (DSB). Our data indicate that pretreatment of prostate cancer cells with HDAC inhibitors (trichostatin A, suberoylanilide hydroxamic acid, MS-275, and OSU-HDAC42) led to increased Ku70 acetylation accompanied by reduced DNA-binding affinity without disrupting the Ku70/Ku80 heterodimer formation. As evidenced by increased Ser(139)-phosphorylated histone H2AX (gammaH2AX), impaired Ku70 function diminished cellular capability to repair DNA DSBs induced by bleomycin, doxorubicin, and etoposide, thereby enhancing their cell-killing effect. This sensitizing effect was most prominent when cells were treated with HDAC inhibitors and DNA-damaging agents sequentially. Mimicking acetylation was done by replacing K282, K317, K331, K338, K539, or K542 with glutamine via site-directed mutagenesis, which combined with computer docking analysis was used to analyze the role of these lysine residues in the interactions of Ku70 with DNA broken ends. Mutagenesis of K282, K338, K539, or K542 suppressed the activity of Ku70 to bind DNA, whereas mutagenesis of K317 or K331 with glutamine had no significant effect. Moreover, overexpression of K282Q or K338Q rendered DU-145 cells more susceptible to the effect of DNA-damaging agents on gammaH2AX formation and cell killing. Overall, the ability of HDAC inhibitors to regulate cellular ability to repair DNA damage by targeting Ku70 acetylation underlies the viability of their combination with DNA-damaging agents as a therapeutic strategy for prostate cancer.