Aim To observe the effects of API0134 on arterial thrombus formation and exploring the mechanism of anti-platelet aggregation. Methods An thrombus model of rabbits with high cholesterolemia was established; and the platelet aggregation function, intracellular ionized calcium i, intracellular ionized magnesium i, cAMP, cGMP and inositol trisphosphate (IP 3) in platelet were examined with Flow cytometer, radioimmunoassay, inositol incoporation and so on. Results The occlusive thrombus formation time was lengthened and the platelet aggregation was inhibited significantly in API0134 group compared with model group. Meanwhile, the increase of platelet concentration of i, i, IP 3 in platelet induced by the thrombus formation were inhibited, and the cAMP concentration in platelet was increased markedly in API0134 treated group compared with control group. Conclusions API0134 exerts strong anti-thrombus and anti-platelet aggregation effects. The mechanism of anti-platelet aggregation is closely related to regulating the balance of intracellular i, i, cAMP and IP 3 in platelet.