Fibrinolytic system impairment contributes to the development of thrombotic disease such as cardiovascular disease and stroke. Therefore, an agent that increases fibrinolytic activity may be useful for the prevention of these diseases. In this study, to explore novel profibrinolytic agents, we examined the profibrinolytic effect of Enzamin, an extract of metabolic products from Bacillus subtilis AK and Lactobacillus in vitro and in vivo. Enzamin directly enhanced plasmin activity generated by tissue-type plasminogen activator (t-PA) by twofold but not by urokinase-type plasminogen activator (u-PA) in vitro, which was measured employing both the chromogenic substrate H-D: -Val-Leu-Lys-pNA (S-2251) and fibrin plate. Enzamin also increased plasmin activity generated by t-PA in the cell lysate and culture medium of endothelial cells, measured by fibrin zymography. Furthermore, the oral administration of a 1% concentration of Enzamin increased plasmin activity generated by t-PA by 1.7-fold but not by u-PA in the euglobulin fraction of mouse plasma. In conclusion, Enzamin has a unique ability to enhance the fibrinolytic activity through an increase in endogenous plasmin activity generated by t-PA released from endothelial cells, and may be a beneficial supplement for the prevention of thrombotic episodes.