Supplemental oxygen therapy (hyperoxia) in preterm babies with respiratory stress is associated with lung injury and the development of bronchopulmonary dysplasia (BPD). Endoplasmic reticulum (ER) homeostasis plays critical roles in maintaining cellular functions such as protein synthesis, folding and secretion. Interruption of ER homeostasis causes ER stress and triggers the unfolded protein response (UPR), which can lead to apoptosis in persistently stressed cells. ERp57 is an ER protein and is associated with calreticulin and calnexin in protein glycosylation. In this study, we found hyperoxia down-regulated ERp57 in neonatal rat lungs and cultured human endothelial cells. Transient transfection of ERp57 siRNA significantly knocked down ERp57 expression and reduced hyperoxia- or tunicamycin-induced apoptosis in human endothelial cells. Apoptosis was decreased from 26.8% to 9.9% in hyperoxia-exposed cells and from 37.8% to 5.0% in tunicamycin-treated cells. The activation of caspse-3 induced by hyperoxia or tunicamycin was diminished and BiP/GRP78 induction was increased in ERp57 knockdown cells. Overexpression of ERp57 exacerbated hyperoxia- or tunicamycin-induced apoptosis in human endothelial cells. Apoptosis was increased from 10.1% to 14.3% in hyperoxia-exposed cells and from 14.0% to 21.2% in tunicamycin-treated cells. Overexpression of ERp57 also augmented tunicamycin-induced caspase-3 activation and reduced BiP/GRP78 induction. Our results demonstrate that ERp57 can regulate apoptosis in human endothelial cells. It appears that knockdown of ERp57 confers cellular protection against hyperoxia- or tunicamycin-induced apoptosis by inhibition of caspase-3 activation and stimulation of BiP/GRP78 induction.