Opening of mitochondrial ATP-sensitive (mitoK_ATP) channel with diazoxide induces an early phase (EP) of cardioprotection. It is unknown whether diazoxide also induces a delayed phase (DP) of cardioprotection. Because nitric oxide (NO) modulates ATP sensitivity of the K_ATP channel, we hypothesized that NO may play a role in diazoxide-induced cardioprotection. Diazoxide (1 mg/kg) was administered either 30 min (for EP) or 24 h (DP) before 30 min of lethal ischemia. Blockers of mitoK_ATP channel [5-hydroxydecanoate (5-HD)] or NO synthase [N ^G-nitro-l-arginine methyl ester (l-NAME)] were given 10 min before ischemia-reperfusion performed by 30 min of left anterior descending coronary artery occlusion and 3 h of reperfusion. A risk area (RA) was demarcated by Evans blue dye, and infarct size (IS) was measured by tetrazolium staining. Diazoxide caused a decrease in IS (%RA) from 27.8 ± 4.2% in the vehicle group to 12.9 ± 1.2% during EP and from 30.4 ± 4.2% in vehicle-treated rabbits to 19.6 ± 2.4% during DP (P < 0.05). IS increased to 31.3 ± 1.1% and 27.9 ± 1.0% (EP) and 29.9 ± 2.3% and 35.1 ± 1.8% (DP) with 5-HD andl-NAME, respectively (P < 0.05). 5-HD andl-NAME caused no proischemic effect in controls. Diazoxide induced both early and delayed anti-ischemic effects via opening of mitoK_ATP channels, which was NO dependent.