Anticoagulation factor Ⅱ (ACFⅡ) from the venom of Agkistrodon acutus forms a 1∶1 complex with activated coagulation factor Ⅹ (FⅩa) in the presence of Ca²⁺ ions. The result that ACFⅡ fails to form a complex with FⅩa in the absence of Ca²⁺ ions indicates that the binding is in the Ca²⁺-dependent manner. ACFⅡ is a new member of the Ⅸ/Ⅹ-bp family, and has a similar amino acid composition to the other members of this family. It is composed of 251 amino acid residues with a relative molecular mass of 29 468.1, determined by MALDI-TOF-MS. ACFⅡ prolongs plasma prothrombin time (PPT) in the dose-dependent fashion and exhibits marked anticoagulant activity only at the concentration higher than the critical concentration (12 nmol/L) of ACFⅡ.