Background: Vascular smooth-muscle cell proliferation plays an important role in atherosclerosis and restenosis. Rhein is an active component extracted from rhubarb. In this study, rhein was found to exert potent inhibitory effects against tumor necrosis factor (TNF)-α-induced human aortic smooth-muscle cells (HASMCs) proliferation. Method: These effects were associated with induced apoptosis, including the induction of Annexin V-positive cells, the cleavage of poly(ADP-ribose)polymerase (PARP), and caspases 3, 8 and 9. Results: Inhibitors of caspases 3, 8 and 9 were efficiently blocked by rhein-induced apoptosis in TNF-α-treated HASMCs. In addition, treatment with rhein resulted in the release of cytochrome c into the cytosol, a loss of mitochondrial membrane potential (ΔΨ<sub>m</sub>), a decrease in Bcl-2 and Bcl-xL and an increase in Bax and Bak expression. However, rhein-mediated apoptosis was blocked by a mitochondrial membrane depolarization inhibitor. These findings indicate that rhein-induced apoptosis occurred via a mitochondrial pathway. Furthermore, the inhibition of mitochondrial membrane depolarization was efficiently blocked by rhein-induced caspase-9 activity, which indicates that the rhein-induced caspase activation signal was downstream of the mitochondrial pathway. Taken together, the results of this study show that rhein inhibits TNF-α-induced HASMC proliferation via mitochondria-dependent apoptosis and that rhein has the potential to act as an anti-atherosclerosis agent.