Thrombotic thrombocytopenic purpura (TTP), a potentially fatal blood clot disorder, is primarily caused by severe deficiency of plasma ADAMTS13 activity resulting from acquired autoantibodies. Plasma exchange is the only effective initial therapy. However, the high mortality rate and the complications associated with plasma exchange therapy remain a major concern. To address unmet clinical needs, therapeutic efficacies of anfibatide, a snake venom-derived platelet glycoprotein Ib antagonist, in murine models of spontaneous thrombocytopenia and shigatoxin-induced TTP were determined. A light scattering platelet aggregometry, microfluidic shear-based assay, and murine models of TTP were used in the study. We showed that purified anfibatide inhibits ristocetin- or botrocetin-induced human or murine platelet agglutination in the presence of von Willebrand factor in a concentration-dependent manner. Anfibatide could also dramatically inhibit the adhesion and aggregation of murine and human platelets on a collagen surface under arterial shear stress, in the presence or absence of plasma ADAMTS13 activity. Most importantly, we demonstrated that an intraperitoneal administration of anfibatide at the dose of 60 ng/g body weight twice daily mitigated spontaneous thrombocytopenia and prevented shigatoxin-induced TTP in Adamts13-/- and disease-susceptible mice (CAST/Ei strain). Thus, we conclude that anfibatide, when administered at the optimal dosage, route, and interval, is efficacious in treating spontaneous and bacterial shigatoxin-induced TTP in the murine models. Our findings may provide the basis for further development of anfibatide for the treatment of acute TTP in humans.