Triptolide, the main active component obtained from Tripterygium wilfordii Hook. f, has been reported to present potent immunosuppressive and anti-inflammatory biological activities. It has been previously shown that due to the cytotoxicity of triptolide it has a limited use in the clinic. Although numerous studies have shown that triptolide induced apoptosis in many human cancer cells there is no report to show triptolide-induced apoptosis in human adrenal cancer cells. We treated the human adrenal cancer NCI-H295 cells with triptolide in vitro and investigated its cytotoxic effects. The cytotoxicity was examined and quantitated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and the viability of inhibition and apoptosis was determined by flow cytometric assay, using propidium iodide (PI) staining for apoptosis. Flow cytometric assay also used for the determination of reactive oxygen species (ROS) production and the levels of mitochondrial membrane potential (ΔΨm), and the caspase-3 and -9 activation in NCI-H295 cells. Western blotting was used for examining the changes of apoptotic associated proteins. The results indicated that triptolide induced cytotoxicity (decreased the percentage of viable cells) and induced sub-G1 phase (apoptosis) occurring in NCI-H295 cells and those effects are dose-dependent. Results also showed that triptolide promoted the production of ROS and decreased the levels of ΔΨm in examined NCI-H295 cells. The results showed that triptolide promoted the levels of cytochrome c, Apaf-1, AIF, Endo G, caspase-9 and -3 which were analyzed by Western blotting. These results suggest that triptolide is able to induce apoptosis on NCI-H295 cells through the mitochondrial-dependent signal pathway.