The N-myc downstream-regulated gene 2 (NDRG2) is involved in cell apoptosis and survival. Although reported to be highly expressed in the cardiac tissue, the biological function of NDRG2 in the heart remains to be established. Insulin exerts protective effects against myocardial ischemia/reperfusion (I/R) injury through the PI3K/Akt pathway. Here, we examined the changes in phosphorylation of NDRG2, a novel substrate and phosphoprotein of Akt, in insulin-induced protection against myocardial I/R. Rat hearts were subjected to 30 min regional ischemia followed by reperfusion with or without insulin at the onset of reperfusion. Reperfusion with insulin inhibited myocardial apoptosis and reduced infarct size, as well as significantly up-regulated myocardial Akt and NDRG2 phosphorylation levels compared with the I/R group. These effects of insulin were blocked by pretreatment with the PI3K inhibitor wortmannin or Akt inhibitor. To further ascertain the role of NDRG2 in insulin-induced cardioprotection, cardiomyocytes were transduced with a lentivirus encoding shRNA targeting NDRG2 (loss-of-function), which rendered the cells more susceptible to I/R injury and significantly blunted the anti-apoptotic effect of insulin. Moreover, the NDRG2 shRNA lentivirus was tested in vivo, and NDRG2 knockdown aggravated myocardial I/R injury and attenuated the insulin-mediated cardioprotection against I/R injury. Taken together, these results suggest a novel role of PI3K/Akt/NDRG2 signaling in the cardioprotective effect of insulin.