Sildenafil induces powerful cardioprotection against ischemia/reperfusion (I/R) injury. Since adenosine is known to be a major trigger of ischemic preconditioning, we hypothesized that A₁ adenosine receptor (A₁AR) activation plays a role in sildenafil-induced cardioprotective signaling. Adult male C57BL wild-type (WT) mice or their corresponding A₁AR knockout (A₁AR-KO) mice were treated intraperitoneally (i.p.) with either sildenafil (0.71 mg/kg, equivalent to 50 mg dose for a 70 kg patient) or volume-matched saline. The selective A₁AR antagonist 8-cyclopentyl-1,3-dipropyxanthine (DPCPX; 0.1 mg/kg, i.p.) was administered 30 min before sildenafil. The hearts were isolated 24 h later and subjected to 30 min of global ischemia and 1 h of reperfusion in Langendorff mode. Post-ischemic myocardial infarct size (mean ± SEM; % of risk area) was reduced in C57BL-WT mice treated with sildenafil (5.6 ± 0.9) versus saline control group (27.3 ± 2.1; p < 0.05; n = 6/each). However, sildenafil failed to protect the A₁AR-KO hearts (31.6 ± 1.9 vs. 32.3 ± 1.5 with saline, p > 0.05). Additionally, DPCPX treatment abolished the infarct limiting effect of sildenafil (27.3 ± 3.2, p < 0.05). DPCPX alone had no effect on infarct size as compared with the control group. No significant changes in post-ischemic recovery of left ventricular pressure and heart rate were observed in the sildenafil-treated group. We further examined the effect of sildenafil in protection against simulated ischemia and reoxygenation injury in adult cardiomyocytes derived from WT and A₁AR-KO mice. WT myocytes treated with sildenafil (1 μM) demonstrated significantly lower trypan blue-positive necrotic cells. However, cardiomyocytes derived from A₁AR-KO mice or DPCPX-treated WT cells failed to show protection against necrosis with sildenafil. These results suggest that A₁AR activation following treatment with sildenafil plays an integral role in the signaling cascade responsible for delayed protection against global I/R injury.