Various proapoptotic stimuli increase the production of superoxide and H 2 O 2 by mitochondria. Whereas superoxide impairs mitochondrial function and is removed by Mn 2+ -dependent superoxide dismutase, the role and metabolism of mitochondrial H 2 O 2 during apoptosis have remained unclear. The effects on apoptotic signaling of depletion of peroxiredoxin (Prx) III, a mitochondrion-specific H 2 O 2 -scavenging enzyme, have now been investigated by RNA interference in HeLa cells. Depletion of Prx III resulted in increased intracellular levels of H 2 O 2 and sensitized cells to induction of apoptosis by staurosporine or TNF-α. The rates of mitochondrial membrane potential collapse, cytochrome c release, and caspase activation were increased in Prx III-depleted cells, and these effects were reversed by ectopic expression of Prx III or mitochondrion-targeted catalase. Depletion of Prx III also exacerbated damage to mitochondrial macromolecules induced by the proapoptotic stimuli. Our results suggest that Prx III is a critical regulator of the abundance of mitochondrial H 2 O 2 , which itself promotes apoptosis in cooperation with other mediators of apoptotic signaling.