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 tissue factor mRNA expression, which resulted in prolonged plasma clotting time, decreased Factor Xa activation, and reduced thrombin generation. Moreover, P-2-CG inhibited thrombosis efficiently by blocking intercellular adhesion molecule 1 and vascular cell adhesion protein 1 expression via tyrosine phosphorylation of nuclear factor-κB p65. P-2-CG inhibits thrombin mediated inflammation and provides a potential therapeutic option for treating endothelial dysfunction and thrombosis.