Diabetic hearts are more vulnerable to ischemia/reperfusion (I/R) injury and less responsive to remifentanil preconditioning (RPC), but the underlying mechanisms are incompletely understood. Caveolin-3 (Cav-3), the dominant isoform of cardiomyocyte caveolae, is reduced in diabetic hearts in which oxidative stress is increased. This study determined whether the compromised RPC in diabetes was an independent manifestation of hyperglycemia-induced oxidative stress or linked to impaired Cav-3 expression with associated signaling abnormality. RPC significantly attenuated postischemic infarction, cardiac dysfunction, myocardial apoptosis, and 15-F2t-isoprostane production (a specific marker of oxidative stress), accompanied with increased Cav-3 expression and enhanced Akt and STAT3 activation in control but not in diabetic rats. Pretreatment with the antioxidant N-acetylcysteine (NAC) attenuated hyperglycemia-induced reduction of Cav-3 expression and Akt and STAT3 activation and restored RPC-mediated cardioprotection in diabetes, which was abolished by cardiac-specific knockdown of Cav-3 by AAV9-shRNA-Cav-3, PI3K/Akt inhibitor wortmannin, or JAK2/STAT3 inhibitor AG490, respectively. Similarly, NAC could restore RPC protection from high glucose and hypoxia/reoxygenation-induced injury evidenced by decreased levels of LDH release, 15-F2t-isoprostane, O₂^-, and JC-1 monomeric cells, which were reversed by caveolae disrupter methyl-β-cyclodextrin, wortmannin, or AG490 in isolated primary cardiomyocytes or siRNAs of Cav-3, Akt, or STAT3 in H9C2 cells. Either methyl-β-cyclodextrin or Cav-3 knockdown reduced Akt and STAT3 activation. Further, the inhibition of Akt activation by a selective inhibitor or siRNA reduced STAT3 activation and vice versa, but they had no effects on Cav-3 expression. Thus, hyperglycemia-induced oxidative stress abrogates RPC cardioprotection by impairing Cav-3-modulated PI3K/Akt and JAK2/STAT3 signaling. Antioxidant treatment with NAC could restore RPC-induced cardioprotection in diabetes by improving Cav-3-dependent Akt and STAT3 activation and by facilitating the cross talk between PI3K/Akt and JAK2/STAT3 signaling pathways.