Type-2 diabetes is caused by insulin resistance, together with apoptosis of beta-cells. High glucose concentrations induce apoptosis of islet cells in vitro, requiring the pro-apoptotic BH3-only proteins Bim and Puma. Glucose-induced production of reactive oxygen species may also activate the NLRP3-inflammasome pathway leading to production of IL-1b. We studied apoptosis activation in response to ER and oxidative stress. Culture of islets in 33.3 mM glucose or 50 mM ribose (a reducing sugar similar to glucose) increased CHOP gene and protein expression, JNK phosphorylation and XBP1 splicing but did not increase mRNA or protein of ER chaperones in wild type islets, indicating activation of pro-apoptotic ER stress signaling. CHOP deficiency partially protected islets from ribose and glucose toxicity. The ER stress inhibitor TUDCA prevented ribose-induced upregulation of CHOP, ATF4 and Puma mRNA. Inhibition of ER stress partially protected wild-type islets, and further protected Bim^-/- or Puma^-/- islets from ribose and glucose toxicity. Loss of Bim or Puma partially protected islets from thapsigargin toxicity. These data indicate that ER stress is the major pathway activated by glucose toxicity, and Bim and Puma are downstream of ER stress signaling in islets. The antioxidant NAC partially protected wild-type islets from ribose-induced apoptosis, but no additional protection was seen in Bim^-/- or Puma^-/- islets. Islets deficient in Bim or Puma were not protected from H₂O₂-induced killing, indicating that Bcl-2-regulated apoptosis is not activated by oxidative stress. Islet macrophage depletion resulted in partial protection from ribose toxicity, suggesting macrophages are involved. However, loss of NLRP3, Caspase1 or IL-1 receptors did not protect islets from ribose, thapsigargin or H₂O₂ toxicity, suggesting that the involvement of macrophages is not through NLRP3 inflammasome activation. Our data suggest that ER and oxidative stress are both involved in glucose-induced killing of islet cells, although different cell death pathways may be activated by these stimuli.