Objective:To analyze and identify differentially expressed phosphoproteins associated with mitochondrial KATP channel opening. Methods: Adult rat ventricular myocytes were isolated, cultured,and identified, and pretreated without or with 100μmol/L diazoxide for 10 min. Phosphoproteins prepared and enriched from the control and diazoxide-pretreated cells were separated by two-dimensional gel electrophoresis (2-DE) followed by sliver staining. The obtained interesting phosphoproteins were further identified by mass spectrometry. Results: Associated with diazoxide preconditioning, the proteins of chaperonin containing TCP-1 and hypothetical protein XP_ 346548 were phosphorylated significantly (P＜0. 01), while the 94-kDa glucose-regulated protein, calpactin I heavy chain and ferritin were dephosphorylated markedly (P＜0. 01). Conclusion: These findings suggest that cardiomyocytes undergo significant posttranslational modification via phosphorylation in a multitude of proteins in order to respond diazoxide preconditioning, and these phosphorylated protein may mediate the downstream signaling of cardioprotection by mitochondrial KATP channel opening induced by ischemic preconditioning.