In this study, we determined the changes in the intracellular redox environment of the heart during ischemia and reperfusion and the effects of resveratrol on such changes. Because redox regulation by thioredoxin (Trx) plays a crucial role in signal transduction and cytoprotection against ROS, the effects of resveratrol on the changes in the amounts of thioredoxin were monitored in an attempt to determine the role of intracellular thioredoxin in resveratrol-mediated changes in intracellular redox environment and its role in resveratrol-mediated cardioprotection. Rats were randomly divided into four groups: group I, control (rats were gavaged with vehicle only); group II, rats were gavaged with 2.5 mg/kg body wt resveratrol per day for 10 days; group III, rats were given resveratrol for 10 days, but on the 7th day, they were treated with shRNA against Trx-1; group IV, rats were given resveratrol for 10 days, but were injected (iv) with cisplatin (1 mg/kg body wt) on days 1, 3, 5, 7, and 9. In concert, two groups of mice (Dn-Trx-1) and a corresponding wild-type group were also gavaged with 2.5 mg/kg body wt resveratrol for 10 days. After 10 days, isolated rat and mouse hearts perfused via working mode were made globally ischemic for 30 min followed by 2 h of reperfusion. Ischemia/reperfusion developed an infarct size of about 40% and resulted in about 25% apoptotic cardiomyocytes, which were reduced by resveratrol. Cisplatin, but not shRNA-Trx-1, abolished the cardioprotective abilities of resveratrol. In the experiments with mouse hearts, similar to rat hearts, resveratrol significantly reduced the ischemia/reperfusion-mediated increase in infarct size and apoptosis in both groups. MDA formation, a presumptive marker for lipid peroxidation, was increased in the I/R group and reduced in the resveratrol group, and resveratrol-mediated reduction in MDA formation was abolished with cisplatin, but not with shRNA-Trx-1. I/R-induced reduction in GSH/GSSH ratio was prevented by resveratrol, and resveratrol-mediated preservation of GSH/GSSG ratio was reduced by cisplatin, but not by sh-RNA-Trx-1. RT-PCR revealed an increase in both Trx-1 and Trx-2 transcripts; but only Trx-2 protein, not Trx-1 protein, was enhanced with resveratrol by Western blot analysis. Electron paramagnetic resonance spectroscopic study revealed that resveratrol treatment significantly increased the decay rates of nitroxide radicals compared to control hearts, suggesting that resveratrol can switch into the reduction state more compared to control heart. Finally, resveratrol generated a survival signal by phosphorylation of Akt and increase in induction of Bcl-2 expression, which was inhibited by cisplatin, but not by shRNA-Trx-1. Taken together, the results of this study indicate that resveratrol provides cardioprotection by maintaining intracellular redox environments, and Trx-2 is likely to play a role in switching I/R-induced death signal into survival signal.