<b><i>Objectives:</i></b> We investigated whether ischemic preconditioning (IPC) protects the right ventricular (RV) myocardium against ischemic injury in hearts treated with the specific mitochondrial ATP-sensitive potassium (K_ATP) channel blocker 5-hydroxydecanoate (5-HD). <b><i>Methods:</i></b> Hearts from male Wistar rats (300 g, n = 39) were isolated and perfused with Krebs-Henseleit buffer and randomized to no IPC (control, n = 16), IPC (n = 16) or IPC preceded by addition of 5-HD (100 µ<smlcap>m</smlcap>, n = 7). IPC consisted of 2 × 5 min of global ischemia followed by 40 min of global ischemia and 120 min of reperfusion. The effect of IPC on RV myocardial infarct size was evaluated by measurement of the infarct size/area-at-risk ratio (RVIS/AAR). Postischemic RV function was evaluated by RV pressures. <b><i>Results:</i></b> IPC produced a marked decrease in RVIS/AAR (24.4 ± 8.1 vs. 42.6 ± 10.6%, p < 0.0001) and improved hemodynamic recovery of RV contractile function compared with the control group. We found no difference in RVIS/AAR (45.2 ± 4.4 vs. 42.6 ± 10.6, p > 0.05) or hemodynamic recovery between IPC + 5-HD and control hearts. Blockade of mitochondrial K_ATP channels by 5-HD abolished the cardioprotective response to IPC. <b><i>Conclusion:</i></b> IPC reduces RV myocardial infarct size and improves postischemic RV contractile function in the isolated rat heart, possibly through opening of the mitochondrial K_ATP channel.