Store-operated Ca²⁺ entry (SOCE) has recently been proposed to contribute to Ca²⁺ influx in vascular smooth muscle cells (VSMCs). Adenosine is known for its protective role against hypoxia and ischemia by increasing nutrient and oxygen supply through vasodilation. This study was designed to examine the hypothesis that SOCE have a functional role in adenosine-induced vasodilation. Small mesenteric resistance arteries and mesenteric VSMCs were obtained from rats. Isometric tensions of isolated artery rings were measured by a sensitive myograph system. Laser scanning confocal microscopy was used to determine the intracellular Ca²⁺ concentration of Fluo-3-loaded VSMCs. Adenosine (0.1-100 µM) relaxed artery rings that were precontracted by phenylephrine in a concentration-dependent manner. In cultured mesenteric VSMCs, passive store depletion by thapsigargin and active store depletion by phenylephrine both induced Ca²⁺ influx due to SOCE. Adenosine inhibited SOCE-mediated increases in cytosolic Ca²⁺ levels evoked by the emptying of the stores. In isolated artery rings, adenosine inhibited SOCE-induced contractions due to store depletion. A_2A receptor antagonism with SCH58261 and adenylate cyclase inhibition with SQ22536 largely attenuated adenosine response. The cAMP analog 8-Br-cAMP mimicked the effects of adenosine on SOCE. Our results indicate a novel mechanism of vasodilatation by adenosine which involves regulation of SOCE through the cAMP signaling pathway due to activation of adenosine A_2A receptors.