Allyl isothiocyanate (AITC) is one of the major components of mustard. The present study for the first time attempted to evaluate the effect of AITC on platelet aggregation. In the in vitro study using platelet-rich plasma (PRP) from rats and humans, AITC at concentrations of 100 and 300 μM significantly inhibited platelet aggregation induced by collagen, thrombin, ADP, and arachidonic acid. AITC also attenuated thromboxane A2 production and ATP release in rat and human PRP. AITC elicited inhibitory effects on cellular Ca(2+) increase and platelet shape change in rat PRP. AITC further showed inhibitory effects on the phosphorylation of PKCδ, p38, ERK, and Akt in rat PRP. In the rat ex vivo study, 1 and 3 mg/kg (po) of AITC showed significant inhibitory effect on platelet aggregation. Furthermore, AITC showed a protective effect in thromboembolism attack model in mouse. These results suggest that AITC has remarkable antiplatelet effects and maybe a therapeutic potential for the prevention of aberrant platelet activation-related disorders.