DNA mismatch repair (MMR) plays a crucial role in maintaining the integrity of the human genome, and impairments in this DNA repair mechanism are responsible for more than 10% of all malignant tumors. Thus, developing a strategy for stage-independent killing of MMR-deficient cells is of great significance in cancer therapy. Here, we show that transient transfection of artificial DNA constructs results in efficient killing of MMR-deficient cancer cells. These constructs were designed so that an intact suicide gene is formed and expressed in the cells, depending on a functional defect in MMR that inhibits single-strand annealing-mediated DNA recombination. In vivo anticancer activity was confirmed in HCT116 xenograft tumor models. Our findings suggest that a cellular DNA recombination-dependent suicide gene can provide a novel tool to selectively kill MMR-deficient tumors, including those resistant to immunotherapy.