Introduction: We have previously shown that over expression of the valosin-containing protein (VCP) in a cardiac specific transgenic (TG) mouse conferred a cardiac protection against ischemia as well as a dose dependent induction of inducible isoform of nitric oxide synthase (iNOS). However, the link between iNOS and the VCP-conferred cardiac protection remains unclear. Hypothesis: Our hypothesis is that VCP elicits cardiac protection by the induction of inducible isoform of nitric oxide synthase (iNOS), leading to the nitrosylation of mitochondrial proteins, resulting in the stimulation of mitochondrial respiration. Methods and Results . Biotin switch was used to identify S-nitrosylated proteins in the VCP TG mouse heart, which a majority of them were found to be mitochondrial proteins involved in the regulation of mitochondrial metabolism and respiration, including succinate semialdehyde dehydrogenase, mitochondrial heat shock protein 60, cytochrome c1. One of the most upregulated nitrosylated proteins was Malic enzyme 3 (Me3), an enzyme which catalyzes the oxidative decarboxylation of malate to pyruvate, was significantly increased by 2.5 fold (p<0.01) in TG mouse compared to WT, although the total protein level was unchanged. Reciprocally, we found a significant decrease by 50% of S-nitrosylated complex I subunit, (NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 9 (NDUFA9)), in VCP TG heart compared to WT (p<0.05). Since S-nitrosylation serves to inhibit Complex I activity, this reduction indicates a reversal of this inhibition by VCP. Basal cardiac mitochondrial respiration was measured using a Clark type oxygen electrode on VCP TG mice (n = 5) and wild-type (WT) mice (n = 6). Mitochondrial State 3 respiration was significantly (p < 0.01) elevated in the VCP TG (72 ±2 nmol O2/min/mg prot) mice compared to WT (45.9 ±6 nmol O2/min/mg prot) with no difference in State 4 or State 2, indicating that VCP over expression enhances the capacity for ATP production without a change in basal levels of respiration. Conclusion: In conclusion, our data reveals a critical and novel role of VCP in mediating cardioprotection through the stimulation of mitochondrial function by inducing nitrosylation of mitochondrial proteins via increased expression of iNOS.