Cordycepin (3′-deoxyadenosine), which comes from Cordyceps militaris, the Chinese medicinal fungal genus Cordyceps, is known to have anti-tumour activity. In this study, we investigated the novel effect of cordycepin on human platelet aggregation that was induced by U46619, a thromboxane A2 (TXA2) analogue. TXA2 is an aggregation-inducing autacoidal molecule that is produced in various agonist-activated platelets. Cordycepin completely inhibited U46619-induced platelet aggregation and simultaneously reduced cytosolic free Ca2+ ([Ca2+]i), which was increased by U46619 (5 μM) up to 66%. Furthermore, the U46619-stimulated phosphorylation of Ca2+-dependent proteins (20 kDa of a myosin light chain and 47 kDa of pleckstrin) was strongly inhibited by cordycepin. These results suggest that cordycepin may have a beneficial effect on autacoidal TXA2-mediated thrombotic diseases by inhibiting TXA2-induced platelet aggregation via suppression of the Ca2+ level.