An earthworm known for its anti-inflammatory, analgesic, antipyretic, and anticancer effects has been recognized as a folk remedy for its facilitatory effect on blood circulation. After complete heat inactivation of endogenous proteases in the earthworm, L. rubellus, an anticoagulant was purified 2,800-fold through ammonium sulfate fractionation, gel permeation chromatography (Sephacryl S-300, Sephadex G-75 and G-150), and C4 reversed-phase HPLC. This water-extractable and hydrophilic activity was stable under heat (100 o C for 30 min.) and acidic conditions (0.4N HCl), which allowed the anticoagulant to survived in a hostile environment following oral administration on its way into the blood. To discover the biochemical nature of the molecule, the anticoagulant was processed with various hydrolases: trypsin, DNase, RNase, and lysozyme, and the resultant samples were analyzed by an in vitro coagulation test and agarose gel electrophoresis. The anticoagulant turned out to be a relatively homogeneous DNA fragment with a relative size of around 72 base pairs. Since a DNA from this earthworm moderately accelerated the antithrombin III inhibition of thrombin compared to heparin, we expect that it could be used as an alternative antithrombotic agent.