Abstract: To investigate the specific cytotoxicity of the anticancer agent NSC-741909 on the growth of a non-small cell lung cancer ( NSCLC ) cell line primarily resistant to gefitinib, and to explore the role of reactive oxygen species ( ROS ) in the process. Methods: Two NSCLC cell lines with different sensitivities to gefitinib were selected, and the effects of gefitinib on cell growth, and induction of apoptosis induction by NSC-741909 were observed. The fluorescent probe 2',7'-dichlorofluorescein diacetate ( DCFH-DA ) was used to label the intracellular ROS and the intracellular fluorescence intensity was detected using flow cytometry ( FCM ). Jun N-terminal kinase ( JNK ) activation was detected using Western blot analysis. Results: NSC-741909 induced the apoptosis of the NSCLC cells primarily resistant to gefitinib. ROS was detected in this cell line. There was an increase in P-JNK expression, whereas the total JNK protein expression remained unchanged. The MKP1 expression was suppressed. Conclusion: NSC-741909, which suppresses mutant K-Ras expression, induced the apoptosis of NSCLC cells that are primarily resistant to gefitinib. This inhibition was mediated by increased ROS production and subsequent JNK activation.