Background: β-cell death due to endoplasmic reticulum (ER) stress has been regarded as an important pathogenic component of type 2 diabetes. The possibility has been suggested that sulfonylurea, currently being used as one of the main oral hypoglyce-mic agents of type 2 diabetes, increases ER stress, which could lead to sulfonylurea failure. The authors of the present study ex-amined ER stress of β-cells in a glucolipotoxic condition using glyburide (GB) in an environment mimicking type 2 diabetes. Methods: Apoptosis was induced by adding various concentrations of GB (0.001 to 200 μM) to a glucolipotoxic condition using 33 mM glucose, and the effects of varied concentrations of palmitate were evaluated via annexin V staining. The markers of ER stress and pro-apoptotic markers were assessed by Western blotting and semi-quantitative reverse transcription-polymerase chain reaction. Additionally, the anti-apoptotic markers were evaluated. Results: Addition of any concentration of GB in 150 μM palmitate and 33 mM glucose did not increase apoptosis. The expres-sion of phosphorylated eukaryotic initiation factor (eIF-2α) was increased and cleaved caspase 3 was decreased by adding GB to a glucolipotoxic condition. However, other ER stress-associated markers such as Bip-1, X-box binding protein-1, ATF-4 and C/EBP-homologous protein transcription factor and anti-apoptotic markers phosphor-p85 phosphatidylinositol 3-kinase and phosphorylation of Akt did not change significantly. Conclusion: GB did not show further deleterious effects on the degree of apoptosis or ER stress of INS-1 cells in a glucolipotox-ic condition. Increased phosphorylation of eIF-2α may attenuate ER stress for adaptation to increased ER protein load.