Anticoagulation factor II (ACF II) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X (FXa)-binding protein with both anticoagulant and hypotensive activities. The thermodynamics of the binding of alkaline earth metal ions to ACF II and their effects on the stability of ACF II and the binding of ACF II to FXa were investigated by isothermal titration calorimetry, fluorescence, differential scanning calorimetry, and surface plasmon resonance. The binding of ACF II to FXa does not have an absolute requirement for Ca2+. Mg2+, Sr2+, and Ba2+ can induce the binding of ACF II to FXa. The radii of the cations bound in ACF II crucially affect the binding affinity of ACF II for cations and the structural stability of ACF II against guanidine hydrochloride and thermal denaturation, whereas the radii of cations bound in FXa markedly affect the binding affinity between ACF II and FXa. The binding affinities of ACF II for cations and the capacities of metal-induced stabilization of ACF II follow the same trend: Ca2+ > Sr2+ > Ba2+. The metal-induced binding affinities of ACF II for FXa follow the trend Mg2+ > Ca2+ > Sr2+ > Ba2+. Although Mg2+ shows significantly low binding affinity with ACF II, Mg2+ is the most effective to induce the binding of ACF II with FXa. Our observations suggest that in blood the bindings of Ca2+ in two sites of ACF II increase the structural stability of ACF II, but these bindings are not essential for the binding of ACF II with FXa, and that the binding of Mg2+ and Ca2+ to FXa may be essential for the recognition between FXa and ACF II. Like Ca2+, the abundant Mg2+ in blood also plays an important role in the anticoagulation of ACF II.