Objective To investigate the effect of deoxyelephantopin (Deo) on regulation of cell apoptosis pathway by reactive oxygen species (ROS) in human non-small cell lung cancer A549 cells. Methods A549 cells were treated with different concentrations of Deo. Cell counting kit 8 (CCK-8) assay was used to detect the cell viability, 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) staining was used to measure cellular ROS content, and Hoechst 33342 staining was used to detect apoptotic bodies. Cell apoptosis and mitochondrial membrane potential were examined by flow cytometry. Quantitative polymerase chain reaction and Western blotting were used to detect the expression of apoptosis-related proteins. A549 xenograft tumor model in nude mice was used to evaluate the anti-tumor effect of Deo in vivo. Hematoxylin-eosin staining and TUNEL assay were used to observe the necrosis and cell apoptosis in tumor tissues. Results With the increase of Deo concentration (1, 2, 4, 8, 16, 32 μmol/L), the viability of A549 cells showed a decreasing trend. Deo at 10 μmol/L could increase the ROS content in A549 cells, reduce mitochondrial membrane potential and promote cell apoptosis. Deo at 10 and 20 μmol/L promoted the mRNA and protein expression of Bax and caspase-3 and inhibited the mRNA and protein expression of Bcl-2, accompanied by significantly increased protein expression of cytochrome C in the cytoplasm. The volume and weight of transplanted tumor in nude mice were significantly inhibited after 17 d of administration of Deo of 10 and 20 mg·kg^－1·d^－1, and the necrotic area and the number of apoptotic cells in the tumor tissue were increased. Conclusion Deo can induce intracellular ROS production, and then activate the mitochondrial apoptosis pathway and consequently promote the apoptosis of A549 cells.