Activation of calpain has been shown to occur in some contexts of cell injury and to be essential for loss of cell viability. Part of this may be mediated at the mitochondrial level. It has been demonstrated that calpain activity is necessary for the complete discharge of apoptosis-inducing factor from the mitochondrial intermembrane space and can cause the cleavage of full-length Bid to a more potent truncated form (Polster, B. M., Basanez, G., Etxebarria, A., Hardwick, J. M., and Nicholls, D. G. (2005) J. Biol. Chem. 280, 6447-6454). In this study, we identify acyl-CoA-binding protein (ACBP) as playing a critical role in the activation of calpain upon exposure of mitochondria to both full-length Bid and truncated Bid (t-Bid). Suppression of ACBP levels by small interfering RNA inhibited the t-Bid-induced activation of mitochondrial mu-calpain and release of apoptosis-inducing factor from the mitochondrial intermembrane space and the cleavage of full-length Bid to t-Bid. Moreover, ACBP required the presence of the peripheral benzodiazepine receptor (for which ACBP is a ligand) to be retained at the mitochondria, to activate mu-calpain, and to amplify Bid-induced mitochondrial damage.