Xylarinase is a bi-functional fibrinolytic metalloprotease isolated from the culture filtrate of endophytic fungus Xylaria curta which is monomeric with a molecular mass of ∼33.76 kDa. The enzyme displayed both plasmin and tissue plasminogen activator like activity under in vitro conditions. It hydrolyses Aα and Bβ chains of the fibrinogen. Optimal fibrinolytic activity of xylarinase is observed at 35 °C, pH 8. Ca(2+) stimulated the fibrinolytic activity of xylarinase while Fe(2+) and Zn(2+) inhibited suggesting it to be a metalloprotease. The Km and Vmax values of xylarinase were 240.9 μM and 1.10 U/ml for fibrinogen and 246 μM and 1.22 U/ml for fibrin, respectively. Xylarinase was found to prolong the activated partial thromboplastin time and prothrombin time. The N-terminal sequence of xylarinase (SNGPLPGGVVWAG) did not show any homology with previously known fibrinolytic enzymes. Thus xylarinase is a novel fibrinolytic metalloprotease which could be possibly used as a new clot busting enzyme.