Habutobin is a thrombin-like enzyme isolated from the crude venom of Trimeresurus Flavoviridis (the habu snake). We have clarified physicochemical and biological characteristics of habutobin. Habutobin was found to have pleiotropic biological activities such as clotting activity of rabbit fibrinogen, inhibitory activity of platelet aggregation, and stimulatory activity of urokinase release from vascular endothelial cells. In the previous study, recombinat-habutobin (r-habutobin) was produced by the baculoviral expression system from habutobin cDNA. r-habutobin has biological activities such as fibrin forming activity and inhibition of collagen-induced platelet aggregation as native habutobin. It has not been clarified how the structure of habutobin relates to its pleiotropic biological activities, which enable us to develop a reliable antithrombotic drug. We produced four fragments of r-habutobin ( F1, F2, F3 and F4) by the truncation of habutobin cDNA in order to identify the functional domain which is responsible for the anti-platelet action of habutobin. We examined whether the r-habutobin fragments prevent platelet aggregation. The effect of two fragments of r-habutobins ( F2 and F3 ) were tested on the aggregation of washed rabbit platelets. Platelet aggregation and ATP release were measured with a light transmission aggregometer using the method of Luciferin-Luciferase reaction. Upon collagen-stimulation of washed platelets, the effect on conformational change of glycoprotein (GP) IIb/IIIa and on the expression of P-selectin were tested by Flow cytometer (FCM) with PAC-1 (which only binds to activated platelets) and P-selectin antibody. The aggregation of washed platelet by collagen was inhibited by F3 r-habutobin. The percent inhibition by F3 r-habutobin was 22.6 ± 5.99 % (n = 7), and that by F2 r-habutobin was 14.75 ± 13.93 (n = 4). F3 r-habutobin also prolonged the lag time of collagen-induced platelet aggregation and slightly inhibited the collagen-induced ATP release from washed platelet. The expression of PAC-1 and P-selectin was decreased by F3 r-habutobin. FCM analysis revealed that F3 r-habutobin decreased FSC/SSC and that platelet membrane expressions of GP IIb/IIIa and P-selectin were decreased by F3 r-habutobin. These results suggested that the reduction of ATP release, decrease of GP IIb/IIIa and P-selectin expression on washed platelets may lead to inhibit the shape change of cytoskeleton in the presence of F3 r-habutobin. The key structure for the inhibition of the expression of P-selectin and the activation of GP IIb/IIIa might be present in F3 r-habutobin.