Mitochondria fulfill a wide array of functions dedicated to the energetic metabolism as well as the control of cell death. These functions imply that mitochondria can be activated by a variety of signals and can integrate them to trigger a process called mitochondrial membrane permeabilization (MMP), which induces the ultimate events of apoptosis. MMP consists in a sudden increase in the permeability of mitochondrial membrane that results in the release of critical proapoptotic intermembrane space effectors into the cytosol such as cytochrome c, apoptosis-inducing factor (AIF), Smac/Diablo, Endo G, and pro-caspases. In many models of apoptosis, mitochondrial translocation of proteins and/or lipids concomitantly with alterations of the intracellular milieu has been shown to activate MMP. This applies to tumor suppressors of the Bax/Bcl-2 family (Bax, Bad, Bid, Bim), several protein kinases (Akt, ASK1, hexokinase), p53, NF-kappaB, and nuclear orphan receptors such as TR3/Nur77. After mitochondrial membrane association, these proteins target constitutive mitochondrial proteins including the permeability transition pore complex (PTPC), Bcl-X(L), HSP70, and/or the lipid interphase. Subsequently, they switch their vital function into a lethal function to promote membrane permeabilization and protein release. In this review, we will describe some general rules of inter-organelle cross-talk activating MMP and will review selected examples of pro-apoptotic protein translocation. Finally, we will propose new pharmacological strategies to modulate this process in a therapeutic perspective.