Objective: Neither glucose nor FFA alone cause clinically meaningful pancreatic β-cell toxicity, especially in subjects with normal or glucose intolerance. The down- and up-regulations of autophagy under the conditions of glucotoxicity and lipotocicity, respectively, are active in β-cells. We hypothesized that glucotoxicity-induced lipophagic insufficiency causes cytotoxic lipid accumulation resulting in glucolipotoxicty in β-cells. Methods: Cell viability assessed by MTT as well as apoptosis by Anexin-V-FITC/PI, cleaved-caspase-3 and AO/PI staining were performed. For β -cell function, insulin contents and GSIS were done. Using real-time-PCR, mRNAs expressions on lipid synthesis, β-oxidation and lipolysis were investigated. SQSTM1/p62, LC3II, p-PERK, peIF2-α, ATF4, IRE1-α, GRP78 and CHOP were evaluated by western blot. Autophagy suppression and stimulation were conducted using siATG5 or ATG5 vector in along with autophagy inhibitors (wortmannin, 3-MA) and activator (trehalose). Results: Neither 0.03 mM palmiate nor 20 mM glucose alone didn’t change cell viability. However, treatment with palmitate + glucose were significantly toxic to INS-1 cells and primary islets (AO/PI). But apoptosis wasn’t significant in INS-1 cells. Compared to other conditions, insulin contents and GSIS were significantly suppressed in islets. Palmitate-induced lipid was significantly higher in the presence of high glucose than low glucose. The mRNA expressions on lipid homeostasis in palmitate + glucose-treated cells were not significantly different than those in palmitate- or glucose-treated cells alone. Glucose suppressed the autophagy induction and flux in a time- and dose-dependent manner. Autophagy induction and flux were upregulated at 0.03 mM palmitate, but upregulated induction and inhibited flux were observed at > 0.5 mM palmitate. Teatment with 20 mM glucose + 0.03 mM palmitate reduced autophagy induction and flux. Also, UPR markers were reduced in accordance with decreased autophagy. Palmitate-induced triglyceride was significantly increased by siATG5, 3 MA or wortmannin. However, treatment with ATG5 vector or trehalose decreased palmitate-induced TG. Finally, trehalose restored insulin contents and GSIS in both palmitae and glucose treated islets. Conclusion: These findings indicate that lipophagic insufficiency induced by glucotoxicity causes toxic lipid accumulation resulting in β-cell dysfunction.