BACKGROUND: Tryptanthrin (Couroupitine A) is isolated from indigo-bearing traditional Chinese herbal medicines. It has a broad spectrum of pharmacological and biological activities. However, the potential effects of tryptanthrin on platelet function and thrombus formation remain elusive. METHODS: Platelets were harvested from C57BL/6 mice and healthy individuals. Following incubation with tryptanthrin, various platelet functions were assessed. Thrombus formation in the presence of tryptanthrin was evaluated both in vitro, using a BioFlux 200 microfluidic system, and in vivo, through FeCl RESULTS: Tryptanthrin inhibited mouse platelet function and impaired carotid artery and deep venous thrombus formation. Tryptanthrin also inhibited human platelet spreading, aggregation and clot retraction. The signaling pathways related to platelet activation, aggregation, hemostasis, and the fibrin clotting cascade were significantly suppressed in platelets treated with tryptanthrin. Notably, the expression of Gp1bα in platelets was diminished by tryptanthrin. CONCLUSIONS: Tryptanthrin impairs platelet function and thrombus formation.