Mitochondria contribute to various diseases and aging phenotypes. Reactive oxygen species (ROS), mainly formed by the respiratory chain, were long thought to cause these effects by damaging proteins, DNA, and lipids. The emerging understanding that ROS act not only destructively but also as dedicated signaling molecules, and that aging processes are regulated by specific signaling networks has stimulated research on mitochondrial signaling systems and the regulation of mitochondrial ROS metabolism. p66Shc is a lifespan-regulating protein contributing to mitochondrial ROS metabolism and regulating the mitochondrial apoptosis pathway. It was found to participate in aging processes and has been implicated in several pathologies. Considerable progress has been made recently concerning the molecular function of p66Shc. It appears that p66Shc responds to a variety of proapoptotic stimuli by increasing ROS levels in the mitochondrial intermembrane space through an inherent ROS-producing activity, and that this ROS formation might trigger initiation of the mitochondrial apoptosis pathway. In this review, we will discuss the current knowledge on the molecular architecture of the p66Shc protein, its role in ROS metabolism and apoptosis regulation in the mitochondrial intermembrane space, the regulation of its mitochondrial transport, and the molecular mechanisms and interactions involved in these processes.