Excessive nitric oxide (NO) production in cytokine-activated β cells has been implicated in β cell disruption in type 1 diabetes. β cells are very vulnerable to NO-induced apoptosis. However, the mechanism underlying this phenomenon is unclear. Low concentrations of NO that lead to apoptosis apparently do not cause severe DNA damage in mouse MIN6 β cells. CHOP, a C/EBP homologous protein that is induced by endoplasmic reticulum (ER) stress and plays a role in growth arrest and cell death, was induced by a NO donor, S-nitroso-N-acetyl-d,l-penicillamine (SNAP). SNAP increased cytosolic Ca²⁺, and only agents depleting ER Ca²⁺ induced CHOP expression and led to apoptosis, suggesting that NO depletes ER Ca²⁺. Overexpression of calreticulin increased the Ca²⁺ content of ER and afforded protection to cells against NO-mediated apoptosis. Furthermore, pancreatic islets from CHOP knockout mice showed resistance to NO. We conclude that NO depletes ER Ca²⁺, causes ER stress, and leads to apoptosis. Thus, ER Ca²⁺ stores are a new target of NO, and the ER stress pathway is a major mechanism of NO-mediated β cell apoptosis.