Lung cancer is the most common cause of cancer death, approximately 85% of which are non-small cell lung cancer (NSCLC). Here we found that artemether (ART), a natural derivative of artemisinin, significantly inhibits the proliferation of NSCLC cells in a dose- and time-dependent manner. We also demonstrated that high concentration of ART induces apoptosis in NSCLC cells through down-regulating the level of anti-apoptotic protein B-cell lymphoma-2 (Bcl-2), cellular inhibitor of apoptosis protein 1 (cIAP1) and cellular inhibitor of apoptosis protein 2 (cIAP2). While low concentration of ART inhibits the mRNA level of cell cycle related genes including cyclin-dependent kinase 1 (CDK1), cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 6 (CDK6), cyclin A2, cyclin B1 and cyclin D1, leading to cell cycle arrest in NSCLC cells. Moreover, we confirmed that low concentration of ART induces DNA double-stranded breaks (DSBs), as well as promoting cellular senescence in NSCLC cells by up-regulating the mRNA and protein level of p16. Taken together, ART represents a promising new anti-NSCLC drug candidate that could attenuate progression of NSCLC cells in a p53-independent manner through inducing apoptosis, cell cycle arrest and promoting cellular senescence.