<p>Lung cancer is the leading cause of cancer-related mortality worldwide, highlighting the novel therapeutic strategies for the treatment of it. 6-Methoxydihydrosanguinarine (6-MS), a derivative of sanguinarine, has been demonstrated to exert cytotoxic effect against various cancer cells. However, the molecular mechanisms involved in anti-lung carcinoma remain underexplored. The present study aims to explore the cytotoxicity of 6-MS towards non-small cell lung cancer (NSCLC) cells and elucidate the potential molecular mechanisms. The proliferation of NSCLC cells was assessed by CCK-8 and EdU incorporation assay. Cell apoptosis was examined by Annexin V-FITC/propidium iodide (PI) double staining. The protein expression was measured by Western blot analysis. Reactive oxygen species (ROS) production was evaluated using DCFH-DA probe. The findings revealed that 6-MS suppressed the proliferation of NSCLC cells in a dose-dependent manner. Results of flow cytometry indicated that 6-MS caused G2/M phase arrest through downregulating the expression of Cdc25C, cyclinB1 and Cdc2. Meanwhile, 6-MS triggered DNA damage, apoptosis and caspases activation, which was associated with the generation of excessive ROS, and could be partially reversed by the cotreatment with N-acetylcysteine (NAC), a ROS scavenger. Furthermore, 6-MS increased the expression of IRE1, phospho-JNK, CHOP and phospho-c-Jun. Pretreatment with JNK inhibitor SP600125 attenuated 6-MS-indcued viability reduction and cleavage of PARP. Additionally, 6-MS blocked Akt/mTOR pathway and downregulated YAP/TAZ and survivin. Overall, this study first reveals that 6-MS inhibits the growth of NSCLC cells by arresting cell cycle, inducing ROS-mediated apoptosis and activating IRE1/JNK signaling. Our study provides a strong rationale for further investigation of 6-MS as a potential therapeutic agent for NSCLC.</p>