Calpains, Ca²⁺-activated cysteine proteases, are cytosolic enzymes implicated in numerous cellular functions and pathologies. We identified a mitochondrial Ca²⁺-inducible protease that hydrolyzed a calpain substrate (SLLVY-AMC) and was inhibited by active site-directed calpain inhibitors as calpain 10, an atypical calpain lacking domain IV. Immunoblot analysis and activity assays revealed calpain 10 in the mitochondrial outer membrane, intermembrane space, inner membrane, and matrix fractions. Mitochondrial staining was observed when C-terminal GFP-tagged calpain 10 was over-expressed in NIH-3T3 cells and the mitochondrial targeting sequence was localized to the N-terminal 15 amino acids. Marked over-expression of mitochondrial calpain 10 resulted in mitochondrial swelling and autophagy that was blocked by the mitochondrial permeability transition (MPT) inhibitor cyclosporine A. Using isolated mitochondria, Ca²⁺-induced mitochondrial permeability transition (MPT) in RCM was partially decreased by calpain inhibitors. More importantly, Ca²⁺-induced inhibition of Complex I of the electron transport chain was blocked by calpain inhibitors and two Complex I proteins were identified as targets of mitochondrial calpain 10, NDUFV2 and ND6. In conclusion, calpain 10 is the first reported mitochondrially-targeted calpain and is a mediator of mitochondrial dysfunction through the cleavage of Complex I subunits and activation of MPT.