Higher requirements for disulfide bond formation in professional secretory cells may affect intracellular redox homeostasis, particularly during an endoplasmic reticulum (ER) stress response. To assess this hypothesis, we investigated the effects of the ER stress response on the major redox couple (GSH/GSSG), endogenous ROS production, expression of genes involved in ER oxidative protein folding, general antioxidant defense, and thiol metabolism using the well validated MIN6 -cell as a model and mouse islets. The data revealed that glucose concentration-dependent decreases in the GSH/GSSG ratio were further decreased significantly by ER-derived oxidative stress induced by inhibiting ER-associated degradation with the specific proteasome inhibitor lactacystin (10 µM) in mouse islets. Notably, minimal cell death was observed during 12 hour treatments. This was likely attributed to the upregulation of genes encoding the rate limiting enzyme for glutathione synthesis (-glutamyl cysteine ligase), as well as genes involved in antioxidant defense (glutathione peroxidase, peroxiredoxin-1) and ER protein folding (Grp78/BiP, PDI, Ero1). Gene expression and reporter assays with a nitric oxide (NO) synthase inhibitor (L-NAME, 1-10 mM) indicated that endogenous NO production was essential for the upregulation of several ER stress responsive genes. Specifically, gel shift analyses demonstrate NO-independent binding of the transcription factor NF-E2-related factor (Nrf2) to the antioxidant response element Gclc-ARE4 in MIN6 cells. However, endogenous NO production was necessary for activation of Gclc-ARE4 driven reporter gene expression. Together these data reveal a distinct protective role for NO during the ER stress response, which helps to dissipate ROS and promote -cell survival.