It has been demonstrated that REV3, the catalytic subunit of the translesion synthesis (TLS) polymerase ζ, play an important role in DNA damage response (DDR) induced by cisplatin, and Ataxia‐telangietasia mutated and Rad‐3‐related (ATR) knase is a central player in activating cell cycle checkpoint, stabilizing replication forks, regulating DDR, and promoting repair of DNA damage caused by cisplatin. Cancer cells deficient in either one of REV3 and ATR are more sensitive to cisplatin. However, whether co‐inhibition of REV3 and ATR can further increase sensitivity of non‐small cell lung cancer (NSCLC) cells to cisplatin is not clear. In this study, we show that REV3 knockdown combined with ATR inhibition further enhance cytotoxicity of cisplatin in NSCLC cells, including cisplatin‐sensitive and ‐resistant cell lines, compared to individual knockdown of REV3 or ATR, which are accompanied by markedly caspase‐dependent apoptosis response, pronounced DNA damage accumulation and severe impediment of interstrand crosslink (ICL), and double strand break (DSB) repair. Our results suggest that REV3 knockdown synergize strongly with ATR inhibition to significantly increase sensitivity of cisplatin in NSCLC cells by inhibiting ICL and DSB repair. Thus simultaneously targeting REV3 and ATR may represent one approach to overcome cisplatin resistance and improve chemotherapeutic efficacy in NSCLC treatment. Herein, we showed that depletion of REV3 combined with ATR inhibition significantly potentiates the cytotoxicity of cisplatin to non‐small cell lung cancer (NSCLC) cells, which is concomitant with pronounced DNA damage accumulation and severe impediment of interstrand crosslink (ICL) and double strand break (DSB) repair, compared to individual depletion of REV3 or ATR. Our data demonstrate that REV3 depletion can synergize with ATR inhibition to increase suppression effect of cisplatin on NSCLC cells through inhibition of ICL and DSB repair.