Factor IX-binding protein (AHP IX-bp), a Ca 2+- and Zn 2+-binding protein from the venom of Agkistrodon Halys Pallas was reported to bind specifically with factor IX in a Zn 2+-dependent manner. Here we have purified AHP IX-bp by a simple two-step of chromatography procedure and found that AHP IX-bp also binds factor Xa (FXa) with high binding-affinity in a Mg 2+-dependent manner. Although Mg 2+ ions have a significantly low binding-affinity for apo-AHP IX-bp as determined by isothermal titration calorimetry, they can induce the binding of apo-AHP IX-bp with FXa even in the absence of Ca 2+ as determined by native PAGE and surface plasmon resonance. Mg 2+ ions are required to maintain in vivo function of FX Gla domain for its recognition of AHP IX-bp. Both Ca 2+ and Zn 2+ ions fail to induce the binding between apo-AHP IX-bp and FXa. The abundant Mg 2+ ions in blood play an important role in the anticoagulation of AHP IX-bp.