This study examined if thioredoxin, the major redox-regulator in the mammalian system, plays any role in the redox signaling of ischemic myocardium. Isolated working rat hearts were made globally ischemic for 30 min followed by 2 h of reperfusion. Another group of hearts was rendered tolerant to ischemia by four cyclic episodes of 5 min ischemia each followed by another 10 min of reperfusion. Reperfusion of ischemic myocardium resulted in the downregulation of thioredoxin 1 (Trx1) expression, which was upregulated in the adapted myocardium. The increased expression of Trx1 was completely blocked with cis-diammine-dichloroplatinum (CDDP), an inhibitor of Trx1. CDDP also abolished cardioprotection afforded by ischemic adaptation as evidenced by a reduction of post-ischemic ventricular recovery, increase in myocardial infarct size and cardiomyocyte apoptosis. The decreased amount of reactive oxygen species in the adapted heart was increased significantly, when Trx1 was blocked with CDDP. The cardioprotective role of Trx1 was further confirmed with transgenic mouse hearts overexpressing Trx1. The Trx1 mouse hearts displayed significantly improved post-ischemic ventricular recovery and reduced myocardial infarct size as compared to the corresponding wild-type mouse hearts. Taken together, the results of this study implicate a crucial role of Trx1 in redox signaling of the ischemic myocardium.