Cryptotanshinone (CTS), a natural product isolated from Salvia miltiorrhiza Bunge, demonstrates anticancer effect. Previous reports showed that CTS induced caspase-independent cell death. Here, we reported that CTS induced pro-death autophagy in human lung cancer cells. CTS inhibited the proliferation of A549 cells in a time- and concentration- dependent manner. CTS triggered autophagy as confirmed by monodansylcadaverine staining, transmission electron microscopy analysis, as well as western blot detection of microtubule-associated protein light-chain 3 (LC3). CTS induced intracellular reactive oxygen species (ROS) formation in a concentration- and time-dependent manner, which was reversed by N-acetyl-L-cysteine (NAC), catalase, diphenyleneiodonium (DPI), pyrrolinodimethylthiocarbamate (PDTC), and dicumarol. Furthermore, CTS-induced autophagy was inhibited by NAC, JNK siRNA and SP600125. NAC reversed CTS-induced JNK phosphorylation. NAC, 3-methyladenine (3-MA), and SP600125 partly reversed CTS-induced cell death. In addition, CTS (10 mg/kg) dramatically inhibited tumor growth by 48.3% in A549 xenograft nude mice, which was completely reversed by NAC (50 mg/kg) co-treatment. Our findings showed that CTS induced pro-death autophagy through activating JNK signaling mediated by increasing intracellular ROS production. © 2016 Bentham Science Publishers.