Mitochondria are known to play a fundamental role in apoptosis by releasing apoptogenic molecules such as cytochrome c into the cytoplasm, thereby sequentially activating initiator caspase‐9. However, the mechanisms of cytochrome c release or caspase‐9 activation in response to hypoxia are unclear. In this report, we show that caspase‐9 is activated by reactive oxygen species (ROS) without involvement of cytochrome c release in hypoxic injury. In addition, activated caspase‐9 induces permeability transition (PT)‐independent cytochrome c release, suggesting that caspase‐9 may disrupt mitochondrial diffusion limit of cytochrome c and serve to amplify further release of cytochrome c.