Human group II secretory phospholipase A2 (hsPLA2) exhibits significant anticoagulant activity that does not require its enzymatic activity. We examined which coagulation factor was targeted by hsPLA2 and analyzed which region of the protein may be involved in this inhibition. Prothrombin time coagulation assays indicated that hsPLA2 did not inhibit activated factor V (FVa) activity, whereas activated factor X (FXa) one-stage coagulation assays suggested that FXa was inhibited. The inhibitory effect of hsPLA2 on prothrombinase activity of FXa, FV, phospholipids, and Ca2+ complex was markedly enhanced upon preincubation of hsPLA2 with FXa but not with FV. Prothrombinase activity was also strongly inhibited by hsPLA2 in the absence of PL. High concentrations of FVa in the prothrombinase generation assay reversed the inhibitory effect of hsPLA2. By using isothermal titration calorimetry, we demonstrated that hsPLA2 binds to FXa in solution with a 1:1 stoichiometry and a Kd of 230 nM. By using surface plasmon resonance we determined the rate constants, kon and koff, of the FXa/hsPLA2 interaction and analyzed the Ca2+ effect on these constants. When preincubated with FXa, synthetic peptides comprising residues 51-74 and 51-62 of hsPLA2 inhibited prothrombinase assays, providing evidence that this part of the molecule, which shares similarities with a region of FVa that binds to FXa, is likely involved in the anticoagulant interaction of hsPLA2 with FXa. In conclusion, we propose that residues 51-62 of hsPLA2 bind to FXa at a FVa-binding site and that hsPLA2 decreases the prothrombinase generation by preventing FXa.FVa complex formation.