Recent developments in the use of small inhibitory RNA molecules (siRNAs) to inhibit specific protein expression have highlighted the potential use of siRNA as a therapeutic agent. The double-strand break signaling/repair proteins ATM, ATR, and DNA-dependent protein kinase catalytic subunit (DNA-PK_cs) are attractive targets to confer enhanced radio and chemosensitivity to tumor cells. We have designed and exogenously delivered plasmids encoding siRNAs targeting these critical kinases to human cancer cells to assess the feasibility of this concept as a clinically translatable experimental therapeutic. siRNA led to a ∼90% reduction in target protein expression. siRNAs targeting ATM and DNA-PK_cs gave rise to a dose-reduction factor of ∼1.4 compared with untransfected and control vector-transfected cells at the clinically relevant radiation doses. This was greater than the radiosensitivity achieved using the phosphatidylinositol 3′-kinase inhibitor Wortmannin or DNA-PK_cs competitive inhibitor LY294002. A similar increased sensitivity to the alkylating agent methyl methanesulfonate (MMS) was also observed for siRNA-mediated ATR silencing. Together, these data provide strong evidence for the potential use of siRNA as a novel radiation/chemotherapy-sensitizing agent.