Tanshinones are a group of bioactive constituents isolated from Salvia miltiorrhiza Bunge, a widely prescribed traditional Chinese herb. In the current study, the anticancer properties of total tanshinones (TDT) were evaluated using 95D lung cancer cells. Tanshinone IIA was identified as the main component of TDT. Compared with tanshinone IIA, TDT showed more cytotoxic effects on the 95D cells. Annexin V/7-AAD double staining, the depolarization of mitochondrial membrane potential (MMP) (Î"Ï), the up-regulation of pro-apoptotic proteins, such as cleaved-PARP, cleaved-caspase-3, Bax, and Bad, and the down-regulation of anti-apoptotic protein Bcl-2 were evidence of TDT-induced apoptosis. Furthermore, TDT-induced autophagy as demonstrated by monodansylcadaverine (MDC) staining and the up-regulation of autophagy-associated proteins, such as LC3-II, Beclin-1, Atg3, Atg5, Atg7, and Atg12. Autophagy inhibitors, 3-methyladenine (3-MA) and bafilomycin A1, enhanced TDT-induced cell death. 3-MA pretreatment enhanced the TDT-induced up-regulation of Bax and cleaved-PARP. In addition, TDT induced the generation of reactive oxygen species (ROS), which was reversed by N-acetylcysteine (NAC). NAC also reversed TDT-induced depolarization of Î"Ï, MDC staining, up-regulation of Bax, cleaved-PARP, Beclin-1, LC3-II, and cell viability. In conclusion, our findings showed that TDT-induced apoptosis and protective autophagy in 95D cells mediated by increasing intracellular ROS production.