Most Kunitz inhibitors exhibit serine protease inhibitory activity, but limited information is available on the regulation of platelet function. Herein, we report the purification and characterization of a novel single Kunitz domain inhibitor (Sibanin) from the salivary glands of the black fly Simulium bannaense. Recombinant Sibanin prolonged activated partial thromboplastin time and prothrombin time, and exhibited high-affinity binding to FXa and elastase with a KD of 5.0 nM and 1.67 nM, respectively. Moreover, Sibanin also shows strong anti-inflammatory and analgesic functions, which would facilitate blood-feeding. Of note, Sibanin markedly suppressed platelet spreading and aggregation, as well as clot retraction. Further studies showed that Sibanin dose-dependently inhibited ADP-induced platelet aggregation by acting on the P2Y12 receptor and blocking its downstream PI3K/AKT/ERK signal pathway. Furthermore, Sibanin also suppressed collagen-induced platelet aggregation by blocking the glycoprotein VI (GPVI) receptor and attenuating the activation of RAP1 signaling pathways. In addition, Sibanin prevented FeCl