Nitric oxide (NO) and protein S-nitrosylation (SNO) have been shown to play important cardioprotective roles in ischemic preconditioning (IPC). Caveolae, a lipid and signaling molecule enriched microdomain of the plasma membrane, have been suggested recently to play an important role in mediating IPC-induced cardioprotection. In Langendorff perfused mouse hearts, infusion of methyl-β-cyclodextrin (MβCD), a cholesterol sequestering agent to disrupt caveolae, before and during four cycles of IPC abolished cardioprotection. In perfusion control hearts, endothelial NO synthase (eNOS) was associated with its inhibitory binding partner caveolin-3. MβCD treatment disrupted caveolae structure and decreased the association of eNOS and caveolin-3. IPC caused activation of AKT/eNOS and downstream NO/SNO signaling. The caveolae-enriched fractions were isolated from mouse hearts by a non-detergent sucrose ultracentrifugation and analyzed by two dimensional DyLight fluorescence difference gel electrophoresis proteomic method for SNO detection. A significant increase of SNO was found in IPC hearts compared to perfusion control, which was blocked by MβCD treatment. Surprisingly the most abundant SNO proteins were mitochondrial proteins. In conclusion, these results suggest an important role of caveolae and caveolin-3-associated eNOS/NO/SNO signaling involved in IPC-mediated cardioprotection.