Abstract: Objective To isolate and identify an anticoagulantion factor, acoagulatin, from the venom of Chinese Agkistrodon and to observe its anticoagulation effect. Method The venom of Chinese Agkistrodon was isolated and purified using ion-exchange chromatography on DEAE-Sepharose Fast Flow and CM-sepharose Fast Flow. High-performance liquid chromatography (HPLC) was performed for determination of the purity of acoagulatin, and SDS-PAGE electrophoresis (with 5% concentrated gel ofpH 6.8, 12% separation gel of pH 8.8, Tris-aminoacetic acid buffer of pH 8.3 as the electrode buffer) for determining the relative molecular mass. For observation of the anticoagulation effect, 20 μ1 acoagulatin solution at the concentration of 0.30, 0.20 and 0.15 μg/μ1, respectively, was mixed with 100 μ1 rabbit anticoagulated plasma and the thrombin time (TT), prothrombin time (PT) and activated partial thromboplastin time (APTT) were determined. Results Acoagulatin was found to consist of two subunits with relative molecular mass of 14 400 and 17 000 respectively, resulting in the total relative molecular mass of 31 400 as determined by SDS-PAGE. HPLC demonstrated good homogeneity of this protein, which significantly prolonged the PT and APTT without affecting TT. Conclusion DEAE-Sepharose Fast Flow and CM-sepharose Fast Flow ion exchange column chromatographies are effective to isolate acoagulatin of high purity, which possesses anticoagulation effect.