This report is designed to explore the exact molecular mechanism by which artesunate (ART), a semisynthetic derivative of the herbal antimalaria drug artemisinin, induces apoptosis in human lung adenocarcinoma (ASTC-a-1 and A549) cell lines. ART treatment induced ROS-mediated apoptosis in a concentration- and time-dependent fashion accompanying the loss of mitochondrial potential and subsequent release of Smac and AIF indicative of intrinsic apoptosis pathway. Blockage of casapse-8 and -9 did not show any inhibitory effect on the ART-induced apoptosis, but which was remarkably prevented by silencing AIF. Of the utmost importance, ART treatment induced the activation of Bak but not Bax, and silencing Bak but not Bax remarkably inhibited ART-induced apoptosis and AIF release. Furthermore, although ART treatment did not induced a significant down-regulation of voltage-dependent anion channel 2 (VDAC2) expression and up-regulation of Bim expression, silencing VDAC2 potently enhanced the ART-induced Bak activation and apoptosis which were significantly prevented by silencing Bim. Collectively, our data firstly demonstrate that ART induces Bak-mediated caspase-independent intrinsic apoptosis in which Bim and VDAC2 as well as AIF play important roles in both ASTC-a-1 and A549 cell lines, indicating a potential therapeutic effect of ART for lung cancer.