CalDAG-GEFI is a guanine nucleotide exchange factor, which actives small GTPase Rap1 and plays an important role in platelet aggregation. Our previous study has shown that CalDAG-GEFI contains redox-sensitive thiols, and its function can be inhibited by thiol modification. In the present study, the effect of CLL2–1, a 1,4-phenanthrenequinone, on CalDAG-GEFI and platelet functions was investigated. In human platelets, CLL2–1 prevented platelet aggregation caused by various stimulators. Flow cytometric analysis revealed that CLL2–1 inhibited GPIIb/IIIa activation and P-selectin secretion. Moreover, CLL2–1 prevented Rap1 activation caused by thrombin, the Ca2+ ionophore A23187, and the diacylglycerol mimetic phorbol 12-myristate 13-acetate, while only slightly inhibited thrombin-induced increases in [Ca2+]i and did not inhibit protein kinase C activation. Western blots after reducing SDS-PAGE showed that treatment of either platelets or platelet lysates with CLL2–1 led to a decrease of monomeric CalDAG-GEFI and appearance of cross-linked oligomers of CalDAG-GEFI, and these effects were inhibited by pretreatment of platelets or lysates with thiol reducing agents prior to the addition of CLL2–1, indicating thiol modification of CalDAG-GEFI by CLL2–1. Furthermore, the thiol reducing agents also prevented the inhibitory effect of CLL2–1 on Rap1 activation, GPIIb/IIIa activation, and platelet aggregation. In CalDAG-GEFI-overexpressing human embryonic kidney 293T cells, CLL2–1 also inhibited CalDAG-GEFI-mediated Rap1 activation. Taken together, our results suggest that the antiplatelet effect of CLL2–1 is due to, at least in part, inhibition of CalDAG-GEFI-mediated Rap1 activation, and provide the basis for development of novel antiplatelet drugs.