Endothelial cell injury underlies an increased occurrence of thromboembolic vascular disease in hereditary hyperhomocysteinemia. We have previously shown that homocysteine causes activation of c-Jun NH2-terminal kinase (JNK) and activating transcription factor 3/liver regenerating factor 1 (ATF3/LRF1) and induces apoptosis in human umbilical vein endothelial cells (HUVECs). In this study, the activation of JNK and ATF3 in HUVECs was mediated by the endoplasmic reticulum. (ER) resident transmembrane kinase IRE1 alpha and beta, which sense and transduce signal of the accumulationj of unfolded proteins in the ER. Moreover, dominant negative mutants of tumor necrosis factor receptor-associated factor 2 and mitogen-activated kinase kinase 4 and 7, as well as antisense ATF3 cDNA, inhibited cell death by homocysteine. These results indicate that the activation of JNK and ATF3 through the ER stress of homocysteine plays a role in the homocysteine-induced cell death. The JNK-ATF3 pathway may be implicated in endothelial cell injury associated with hereditary hyperhomocysteinemia.