To provide further pharmacological evidence for its clinical use in thrombotic diseases, the antithrombotic activities of the aqueous extract of Radix Ophiopogon japonicus (ROJ-ext) were studied in mouse and rat models. The results showed that ROJ-ext remarkably decreased length of tail thrombus in mice at 48 h and 72 h after carrageenan injection at doses of 12.5 and 25.0 mg/kg. Meanwhile, ROJ-ext markedly inhibited thrombosis induced by arterial-venous (AV) shunt (silk thread) in rats at doses of 6.25 and 12.5 mg/kg. Furthermore, ROJ-ext and one of its components, ruscogenin, significantly inhibited platelet aggregation induced by adenosine diphosphate (ADP) in rats by oral administration of 12.5 mg/kg or 0.7 mg/kg for three times, however, ophiopogonin D 1.4 mg/kg only showed slight inhibition. On the other hand, ophiopogonin D (0.5-2.0 mg/kg, p.o.) and ruscogenin (0.25-1.00 mg/kg, p.o.) produced dose-related inhibition of venous thrombosis induced by tight ligation of the inferior vena cava for 6 h in mice by once oral administration. The findings of this study indicate that an aqueous extract of Radix Ophiopogon japonicus (ROJ-ext) exerted significant antithrombotic activity and ruscogenin and ophiopogonin D are two of its active components, which supported its therapeutic use for thrombotic diseases.