Many anticoagulant proteins have been found from snake venoms. Recently, (L)-amino acid oxidase (LAO) from the venom of Gloydius blomhoffi, M-LAO, was reported to inhibit coagulation factor IX; however, the mechanism of its anticoagulant activity is still unclear. Here, we re-evaluated the anticoagulant activity of M-LAO. We first purified M-LAO from the venom of G. blomhoffi, and examined the effect of LAO inhibitors and the hydrogen peroxide scavenger, catalase, on the anticoagulant activity of M-LAO. We found that the isolated M-LAO fraction prolongs the APTT, PT and fibrinogen clotting time and cleaves the Aalpha-chain of fibrinogen. LAO inhibitors or catalase did not inhibit these effects. Detailed analysis revealed that the M-LAO fraction contained a small amount of 39-kDa metalloproteinase. The prolongation of clotting time and degradation of fibrinogen were inhibited by a metalloproteinase inhibitor. Therefore, we concluded that the anticoagulant activity of the M-LAO fraction was caused by the 39-kDa metalloproteinase.