Introduction: Mitochondrial dysfunction plays a critical role in mediating cell death during ischemia/reperfusion (IR). We previously found that overexpressing valosin-containing protein (VCP), an ATPase associate-binding protein, in a transgenic (TG) mouse heart increases the expression of inducible nitric oxide synthase (iNOS) and reduces infarct size during IR. We tested the hypothesis that VCP mediates cardiac protection by increasing mitochondrial respiration and inhibiting the opening of the mitochondrial permeability transition pore (mPTP) via iNOS. Methods and Results: Cardiac mitochondria were isolated from the heart tissues of adult VCP TG and their litter-matched WT mice (N=6/group). VCP TG mice increased the expression of VCP by 3.1 folds and iNOS by 4.5 folds in the mitochondrial fraction vs WT ( p <0.01). Mitochondrial respiration was determined by oxygen consumption rate (OCR) in an airtight chamber with a Clark-type electrode. Compared to WT, mitochondria from VCP TG showed a significant increase in OCR during ADP-dependent State 3 under Complex I stimulation while no significant change was seen in oligomycin induced-State 4, increasing the efficiency of mitochondrial respiration, as measured by the respiratory control ratio (State 3/State 4). VCP TG mice also displayed a significant increase in their maximum respiration capacity versus WT, measured after the addition of the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone. However, there was no difference between VCP TG and WT on Complex II-dependent respiration rate, suggesting VCP’s predominance on Complex I-stimulated respiration. mPTP opening was induced by Ca 2+ overload in isolated mitochondria and was measured by a swelling assay. Compared to WT mice, VCP TG showed significantly less mitochondrial swelling under the Ca 2+ overload ( p <0.05). To test whether protection by VCP is mediated by iNOS, a bigenic VCP TG /iNOS knock out mouse was generated. Deletion of iNOS from the VCP TG abolishes the effects of VCP on mitochondrial respiration and mPTP opening. Conclusion: VCP prevents the opening of the mPTP and increases mitochondrial respiratory efficiency in an iNOS dependent manner, which may represent a novel mechanism of cardiac protection.