Objective: Glucolipotoxicity (GLT) of pancreatic beta cells caused by elevated circulating free fatty acids and high glucose has been recognized to be a crucial factor contributing to beta cell dysfunction and beta cell loss in type 2 diabetes. Many factors and their related signal pathways (oxidative stress, ER stress, disruption in Ca2+ homeostasis etc.) have been reported to be involved in GLT-induced beta cell damage and recent research has shown that mitogen-activated protein kinase (MAPK) signaling pathways are activated in response to GLT-induced cellular stress, however, the overall underlying mechanisms remained poorly understood. Methods: We treated insulin-secreting HIT-T15 pancreatic beta cells with 25 mM glucose and palmitic acid (PA) or oleic acid (OA) mixed with 10% BSA to induce GLT and observed its effect on cell apoptosis by using following methods: Assessment of cell viability: MTT assay. Measurement of apoptosis by DAPI staining and DNA fragmentation by agarose gel electrophoresis. Protein expression was evaluated by Western blot. For additional treatment study, cells were pretreated with either U0126 or AICAR for 1 h and then co-treated with 0.1 mM PA or OA for 16 h. All results are expressed as a mean ± S.D. Statistical analysis was performed using one-way analysis of variance (ANOVA) with a subsequent Tukey’s multiple comparison test. A P value < 0.05 was used as a threshold for statistical significant. Results: PA, but not OA, induced apoptosis in beta cells by activation MAPK family members: ERK, p38, JNK, and the AMPK activator (AICAR) and ERK inhibitor (U0126) reduced GLT-induced apoptosis by inhibiting MAPKs. Also we revealed that ERK is located upstream of p38 and JNK. Conclusion: MAPKs, especially ERK, play a pivotal role in GLT-induced beta cell damage and AMPK activation and ERK inhibition can restrain the activity of this pro-apoptotic pathway.