Carbon monoxide (CO), a reaction product of the cytoprotective heme oxygenase (HO)-1, is antiapoptotic in a variety of models of cellular injury, but the precise mechanisms remain to be established. In human umbilical vein endothelial cells, exogenous CO activated Nrf2 through the phosphorylation of protein kinase R–like endoplasmic reticulum kinase (PERK), resulting in HO-1 expression. CO-induced activation of PERK was followed by the phosphorylation of eukaryotic translation initiation factor 2 and the expression of activating transcription factor 4. However, CO fails to induce X-box binding protein-1 expression and activating transcription factor 6 cleavage. CO had no significant effect on synthesis of endoplasmic reticulum (ER) chaperone proteins such as the 78-kDa glucose-regulated protein 78 and 94. Instead, CO prevented X-box binding protein 1 expression and activating transcription factor 6 cleavage induced by ER-stress inducers such as thapsigargin, tunicamycin and homocysteine. CO also prevented endothelial apoptosis triggered by these ER inducers through suppression of C/EBP homologous protein expression, which was associated with its activation of p38 mitogen-activated protein kinase. Similarly, endogenous CO produced from endothelial HO-1 induced by either exogenous CO or a pharmacological inducer was also cytoprotective against ER stress through C/EBP homologous protein suppression. Our findings suggest that CO renders endothelial cells resistant to ER stress not only by downregulating C/EBP homologous protein expression via p38 mitogen-activated protein kinase activation but also by upregulating Nrf2-dependent HO-1 expression via PERK activation. Thus, the HO-1/CO system might be potential therapeutics in vascular diseases associated with ER stress.