Conflicting results exist regarding the role of A₃ adenosine receptors (A₃ARs) in mediating cardioprotection during reperfusion following myocardial infarction. We hypothesized that the effects of the A₃AR agonist IB-MECA to produce cardioprotection might involve activation of other adenosine receptor subtypes. C57Bl/6 (B6), A₃AR KO, A_2AAR KO, and A_2AAR KO/WT bone marrow chimeric mice were assigned to 12 groups undergoing either hemodynamic studies or 45 min of LAD occlusion and 60 min of reperfusion. IB-MECA (100 μg/kg) or vehicle was administered by iv bolus 5 min before reperfusion. Radioligand binding assays showed that IB-MECA has high affinity for the mouse A₃AR (K _i = 0.17 ± 0.05 nM), but also can bind with lower affinity to the A₁AR (9.0 ± 2.4nM) or the A_2AAR (56.5 ± 10.2nM). IB-MECA caused bi-phasic hemodynamic changes, which were completely absent in A₃AR KO mice and were modified by A_2AAR blockade or deletion. IB-MECA stimulated histamine release, increased heart rate, and significantly reduced IF size in B6 mice from 61.5 ± 1.4 to 48.6 ± 2.4 % of risk region (RR; 21 % reduction, p < 0.05) but not in A₃AR KO mice. Compared to B6, A₃AR KO mice had significantly reduced IF size (p < 0.05). In B6/B6 bone marrow chimeras, IB-MECA caused a 47 % reduction of IF size (from 47.3 ± 3.9 to 24.7 ± 4.5, p < 0.05). However, no significant cardioprotective effect of IB-MECA was observed in A_2AARKO/B6 mice, which lacked A_2AARs only on their bone marrow-derived cells. Activation of A₃ARs induces a bi-phasic hemodynamic response, which is partially mediated by activation of A_2AARs. The cardioprotective effect of IB-MECA is due to the initial activation of A₃AR followed by activation of A_2AARs in bone marrow-derived cells.