The protein kinase encoded by the ataxia telangiectasia and Rad3-related (ATR) gene is activated by DNA-damaging agents that are frequently used as anticancer therapeutics. Inhibition of ATR expression in cultured cancer cells has been demonstrated to increase sensitivity to chemotherapeutic drugs, including the DNA-crosslinking agent cisplatin. Cisplatin is a widely used and effective drug, but its use is associated with significant toxicity. Here, we demonstrate that genetic inhibition of ATR expression selectively enhanced cisplatin sensitivity in human colorectal cancer cells with inactivated p53. A knock-in strategy was used to restore wild-type p53 in cells harboring wild-type or mutant ATR alleles. Knock-in of functional p53 in ATR-deficient cells restored checkpoint function, suppressed apoptotic pathways and markedly increased clonogenic survival after cisplatin treatment. These results suggest that a strategy that combines specific inhibitors of ATR and conventional therapies might promote synthetic lethality in p53-deficient tumors, and thus minimize toxicity to normal tissues.