Oridonin, a diterpenoid isolated from Rabdosia rubescences, could induce apoptosis through the generation of reactive oxygen species (ROS) in human hepatoma HepG2 cells. p53, a specific inhibitor of pifithrin alpha (PFT alpha), markedly inhibited ROS generation and apoptosis, showing that p53 was responsible for the cytotoxity of oridonin through mediation by ROS. Moreover, the ROS activated the p38 kinase, which in turn promoted the activation of p53, as verified by evidence showing that the ROS scavenger N-acetyl-cysteine (NAC) not only blocked the phosphorylation of p38 but also partially inhibited the activation of p53, and the p38 inhibitor SB203580 reduced the activation of p53 as well. Mitochondria were either the sources or the targets of ROS. This study showed that oridonin stimulated mitochondrial transmembrane permeabilization in a ROS-dependent manner because NAC almost thoroughly reversed the drop of mitochondrial transmembrane potential (Deltapsim) and the release of cytochrome c from the mitochondrial inter-membrane space into cytosol. Furthermore, as a result of mitochondrial permeability transition, procaspases-9 and -3 were cleaved into 37- and 17-kDa proteolytic products, respectively, which acted as executors of oridonin-induced apoptosis.