Oxygen-regulated protein 150 (ORP150) is an inducible endoplasmic reticulum (ER) chaperone molecule that is up-regulated after numerous cellular insults and has a cytoprotective role in renal, neural, and cardiac models of ischemia/reperfusion injury. ORP150 also has been shown to play a role in cellular Ca²⁺ homeostasis, and in turn, regulating calpain activity. In this study, we identified ORP150 in whole rat renal cortical mitochondria and matrix fractions, demonstrated the targeting of an ORP150-GFP construct to the mitochondria of NIH-3T3 cells, and showed that the N-terminal 13 amino acids of ORP150 are sufficient for this translocation. ORP150 expression was found to be regulated by the CHOP/GADD153 transcription factor and ORP150 levels increased in the mitochondria and ER of COS-7 cells after diverse stresses including hypoxia, serum starvation, prolyl hydroxylase inhibition with dimethyloxaloylglycine, and exposure to tunicamycin, ethidium bromide, and 2-deoxyglucose. Induction of the mitochondrial specific stress response in COS-7 cells through expression of an ornithine transcarbamylase mutant (OTC) increased mitochondrial ORP150 levels and mitochondrial calpain activity. To determine whether mitochondrial ORP150 and mitochondrial calpain 10 interact, rat cortical mitochondria exposed to Ca²⁺ resulted in ORP150 cleavage in a calpain inhibitor-dependent manner, revealing that ORP150 is a substrate and may be regulated by calpain 10. These data reveal a novel cellular localization for ORP150 and that mitochondrial ORP150 is upregulated by CHOP/GADD153 in response to mitochondrial and ER stress. Our data also reveal that ORP150 is a substrate for mitochondrial calpain 10.