Oridonin (1), an active component isolated from the plant Rabdosia rubescens, has been reported to exhibit antitumor effects. In this study, the mechanism involved in 1-induced growth inhibition, including apoptosis and G(2)/M phase arrest, in human laryngeal carcinoma HEp-2 cells deficient in functional p53, was investigated for the first time. Compound 1 triggered the mitochondrial apoptotic pathway, as indicated by increased Bax/Bcl-2 ratios, reduction of mitochondrial membrane potential (DeltaPsi(m)), and substantial increase in apoptosis-inducing factor (AIF) and cytochrome c. Inhibition of caspase-9 in HEp-2 cells did not protect the cells from 1-induced apoptosis, and cleaved caspase-9 was not detected, indicating that apoptosis occurred via a caspase-9-independent pathway. The results also suggested that G(2)/M phase arrest and apoptosis mediated by 1 occurred via a p53-independent but in a p21/WAF1-dependent manner in HEp-2 cells. In addition, the generation of reactive oxygen species (ROS) was found to be a critical mediator in growth inhibition induced by 1. Taken together, the results indicate that oridonin (1) is a potentially effective agent for the treatment of laryngeal squamous cell carcinoma.