The selection of vascular grafts for coronary artery bypass surgery is crucial for a positive outcome. This study aimed to establish a novel line of vascular endothelial cells with a potent anticoagulant effect. A lentiviral vector was used to stably transfect human umbilical vein endothelial cells (HUVECs) with PGI2S alone (HUVEC-PGI2S) or both PGI2S and tPA (HUVEC-PGI2S-tPA). Both HUVEC-PGI2S and HUVEC-PGI2S-tPA cells over-expressing PGI2S and tPA were compared to mock-transfected cells. The enzyme-linked immuno sorbent assay (ELISAs) demonstrated that the anticoagulation components, ATIII and PLG, were up-regulated and coagulation factor FVIII was down-regulated in both cell lines. QRT-PCR and western blotting demonstrated the vasodilation and platelet disaggregation proteins PKA, PKC, and PTGIR were up-regulated in both cell lines, but MAPK expression was not altered in either cell line. However, cell viability and colony formation assays and cell cycle analysis demonstrated that both cell lines had a lower rate of cell growth and induced G1 phase arrest. HUVEC-PGI2S and HUVEC-PGI2S-tPA cells have a potent anticoagulant effect and their use in vascular heterografts may decrease the risk of thrombosis.