We previously reported that the fibrinogen-binding segment (residues 221-550) of Staphylococcus aureus clumping factor A (ClfA), which binds to fibrinogen gamma chain C-terminus, exerted inhibitory effects on platelet aggregation and fibrin clot formation in vitro. Here, we further demonstrated the effectiveness of using ClfA221-550 to inhibit platelet-rich thrombus formation in vivo. Platelet-rich thrombi were formed in the mesenteric venules of fluorescein-loaded mice by filtered light illumination. It grew rapidly and ultimately resulted in the cessation of blood flow due to vessel occlusion. Given by intravenous bolus injection, ClfA221-550 delayed occlusive thrombi formation in a dose-dependent manner: 2-, 3- and 4.5-fold prolongations of vessel occlusion time were attained with 0.69, 6.9 and 34.5 mg/kg of ClfA221-550, respectively. Reduced fibrin clot formation at the late phase with plasmas, which were prepared from ClfA221-550-treated mice, was also dose-dependent. The suppression of fibrin formation ex vivo coincided with the delay of occlusive thrombus formation in vivo, suggesting that the antithrombotic effect of ClfA221-550 may result from the blockade of fibrinogen gamma chain C-terminal functions, in mediating platelet aggregation and fibrin clot formation. Administration of ClfA221-550 also lengthened the tail bleeding of mice; however, significant effect was achieved only with a higher dosage, namely 34.5 mg/kg. These results together showed that blockade of fibrinogen gamma chain C-terminus with ClfA221-550 preferentially affected platelet-rich thrombus formation rather than normal haemostasis, thus providing a rationale for selecting fibrinogen gamma chain C-terminus as a new target for thrombotic intervention.