Inhibitors of thrombin, a key enzyme in the blood coagulation cascade, are of great interest because of their selective specificity and effectiveness in anticoagulation therapy against cardiovascular disorders. The natural soybean phytosterol, β-sitosterol (BSS) demonstrated anticoagulant activity by dose-dependent inhibition of thrombin in an uncompetitive manner with a Ki value of 0.267 μM as well as by partial inhibition of thrombin-catalyzed platelet aggregation with a half-maximal inhibitory concentration (IC50) value of 10.45 ± 2.88 μM against platelet-rich plasma and 9.2 ± 1.2 μM against washed platelets. An in silico study indicated binding of BSS to thrombin, which was experimentally verified by spectrofluorometric and isothermal calorimetric analyses. Under in vitro conditions, BSS demonstrated thrombolytic activity by activating plasminogen, albeit it is devoid of protease (fibrinogenolytic) activity. BSS was noncytotoxic to mammalian cells, nonhemolytic, demonstrated its in vivo anticoagulant activity when administered orally, and inhibited k-carrageen-induced thrombus formation in the tails of mice. Our results suggest that dietary supplementation of BSS may help to prevent thrombosis-associated cardiovascular disorders.