Prostaglandins are naturally occurring local mediators that can participate in the modulation of the cardiovascular system through their interaction with Gs/Gi-coupled receptors in different tissues and cells, including platelets. Thrombin is one of the most important factors that regulates platelet reactivity and coagulation. Clinical trials have consistently shown that omega-3 fatty acid supplementation lowers the risk for cardiovascular mortality and morbidity. Since omega-3 fatty acids are the main precursors of PGE3 in vivo, it would be relevant to investigate the effects of PGE3 on Thrombin Receptor Activating Peptide (TRAP-6)-induced platelet reactivity to determine the receptors and possible mechanisms of action of these compounds. We have measured platelet aggregation, P-selectin expression, and vasodilator-stimulated phosphoprotein (VASP) phosphorylation to evaluate platelet reactivity induced by TRAP-6 to determine the effects of PGE3 on platelet function. We assessed the ability of DG-041, a selective prostanoid EP3 receptor antagonist, and of ONO-AE3-208, a selective prostanoid EP4 receptor antagonist, to modify the effects of PGE3. PGE3 inhibited TRAP-6-induced platelet aggregation and activation. This inhibition was enhanced in the presence of a Gi-coupled EP3 receptor antagonist and abolished in the presence of a Gs-coupled EP4 receptor antagonist. The effects of PGE3 were directly related to changes in cAMP, assessed by VASP phosphorylation. The general effects of PGE3 on human platelet reactivity are the consequence of a balance between activatory and inhibitory effects at receptors that have contrary effects on adenylate cyclase. These results indicate a potential mechanism by which omega-3 fatty acids underlie cardioprotective effects.