Thrombin blockers have been shown to be effective for various pathological conditions, but their use is limited due to the potential for serious bleeding adverse effects. This study introduced a novel bioactive peptide (P-2-CG) from oyster, that mitigated thrombin-mediated barrier dysfunction and prothrombotic phenotypes in human pulmonary microvascular endothelial cells (HPMECs). P-2-CG significantly attenuated the increase in endothelial monolayer permeability induced by thrombin through the possible attenuation of RhoA activation and it promoted barrier recovery by enhancing endothelial cell adhesion. Additionally, P-2-CG was found to decrease the pro-thrombotic phenotype induced by thrombin in HPMEC by reducing the extrinsic trigger TF mRNA expression, which resulted in prolonged plasma clotting time, decreased FXa activation, and reduced thrombin generation. Moreover, P-2-CG inhibited thrombosis efficiently by blocking ICAM-1 and VCAM-1 expression via tyrosine phosphorylation of RelA/p65. P-2-CG inhibits thrombin mediated inflammation and provides a potential therapeutic option for treating endothelial dysfunction and thrombosis.