Adenosine exerts a potent cardioprotective effect that is mediated by adenosine A1 and A3 receptors. The signaling pathways activated by the A1 and A3 receptors are distinct and involve selective coupling to phospholipases C and D, respectively. The objective of our study was to elucidate the signaling mechanism that mediates the coupling of each receptor to its respective phospholipase and to test the role of RhoA as a novel mediator leading from adenosine receptors to cardioprotection. C3 transferase and dominant negative RhoA (RhoAT19N) blocked the A3 receptor-mediated phospholipase D activation and cardioprotection but had no effect on A1 receptor-mediated phospholipase C activation or cardioprotection. In contrast, pertussis toxin treatment caused a greater inhibition of the diacylglycerol accumulation induced by the A1 agonist than by the A3 agonist, and it completely abrogated the A1 agonist-mediated cardioprotection. Thus, adenosine A1 and A3 receptors are linked to different G-proteins. The A3 receptor is coupled via RhoA to activate phospholipase D in exerting its cardioprotective effect, whereas the A1 receptor is linked via Gi to phospholipase C to produce cardioprotective responses. The present study identifies a novel role for RhoA and further suggests its importance in regulating cardiac cellular function.