We have cloned an earthworm-derived Factor Xa (FXa) inhibitor, with an excellent inhibitory specificity from the midgut of the Eisenia andrei. We designate this inhibitor eisenstasin. An eisenstasin-derived small peptide (ESP) was synthesized and we examined whether ESP played an essential role in FXa inhibition. Compared to antistasin-derived small peptides (ASP) originating from leech, ESP primarily exhibited a high level of FXa inhibition in chromogenic peptide substrate assays and revealed an approximately 2-fold greater inhibition of FXa cleavage of a target protein than ASP. This suggests that ESP could be an effective anti-coagulant that targets FXa during the propagation step of coagulation. ESP also inhibited proteinase-activated receptor 2-mediated FXa activation, which may trigger endothelial inflammation. Endothelial nitric oxide (NO) was significantly reduced by ESP (p<0.0001), indicating that protease-activated receptor-2 (PAR-2) was effectively inactivated. We also found that ESP reduced the expressions of pro-inflammatory cytokines (IL-1alpha, IL-1beta, IL-8, IL-16, MCP-1, MIP-1alpha and MIP-1beta) by cultured cells treated with both ESP and FXa. Our results provide the first evidence that ESP might interrupt coagulation cascades by inhibiting FXa, and thereby may effectively control the bidirectional alternation between coagulation and inflammation.