Activation of adenosine A1 or A3 receptors protects heart cells from ischemia‐induced injury. The A₃ receptor signals via RhoA and phospholipase D (PLD) to induce cardioprotection. The objective of the study was to investigate how RhoA activates PLD to achieve the anti‐ischemic effect of adenosine A3 receptors. In an established cardiac myocyte model of preconditioning using the cultured chick embryo heart cells, overexpression of the RhoA‐noninteracting PLD1 mutant I870R selectively blocked the A3 agonist (Cl‐IBMECA, 10 nM)‐induced cardioprotection. I870R caused a significantly higher percentage of cardiac cells killed in A₃ agonist‐treated than in A₁ agonist (CCPA, 10 nM)‐treated myocytes (ANOVA and posttest comparison, P<0.01). Consistent with its inhibitory effect on the PLD activity, I870R attenuated the Cl‐IBMECA‐mediated PLD activation. Cl‐IBMECA caused a 41 ± 15% increase in PLD activity in mock‐transfected myocytes (P<0.01, paired t test) while having only a slight stimulatory effect on the PLD activity in I870R‐transfected cells. To further test the anti‐ischemic role of a direct RhoA‐PLD1 interaction, atrial cardiac myocytes were rendered null for native adenosine receptors by treatment with irreversible A₁ antagonist m‐DITC‐XAC and were selectively transfected with the human adenosine A₁ or A₃ receptor cDNA individually or they were cotransfected with cDNAs encoding either receptor plus I870R. I870R preferentially inhibited the human A₃ receptor‐mediated protection from ischemia. The RhoA‐noninteracting PLD1 mutant caused a significantly higher percentage of cardiac cells killed in myocytes cotransfected with the human A₃ receptor than in those cells expressing the human A₁ receptor (ANOVA and posttest comparison, P<0.01). The present data provided the first demonstration of a novel physiological role for the direct RhoA‐PLD1 interaction, that of potent protection from cardiac ischemia. The study further supported the concept that a divergent signaling mechanism mediates the anti‐ischemic effect of adenosine A₁ and A₃ receptors.