Stigmalactam, an aristolactam-type alkaloid extracted from Orophea enterocarpa, exerts cytotoxicityagainst several human and murine cancer cell lines, but the molecular mechanisms remain elusive. The aimsof this study were to identify the mode and mechanisms of human cancer cell death induced by stigmalactamemploying human hepatocellular carcinoma HepG2 and human invasive breast cancer MDA-MB-231 cellsas models, compared to normal murine fibroblasts. It was found that stigmalactam was toxic to HepG2 andMDA-MB-231 cells with IC50 levels of 23.0±2.67 μM and 33.2±4.54 μM, respectively, using MTT assays. At thesame time the IC50 level towards murine normal fibroblast NIH3T3 cells was 24.4±6.75 μM. Reactive oxygenspecies (ROS) production was reduced in stigmalactam-treated cells dose dependently after 4 h of incubation,indicating antioxidant activity, measured by using 2’,7’,-dichlorohydrofluorescein diacetate and flow cytometry.Caspase-3 and caspase-9 activities were increased in a dose response manner, while stigmalactam decreased themitochondrial transmembrane potential dose-dependently in HepG2 cells, using 3,3’-dihexyloxacarbocyanineiodide and flow cytometry, indicating mitochondrial pathway-mediated apoptosis. In conclusion, stigmalactamfrom O. enterocarpa was toxic to both HepG2 and MDA-MB-231 cells and induced human cancer HepG2 cellsto undergo apoptosis via the intrinsic (mitochondrial) pathway.