Pancreatic endocrine cells employ a sophisticated system of paracrine and autocrine signals to synchronize their activities, including glutamate which controls hormone release and β-cell viability by acting on glutamate receptors. The glutamate transporter EAAT2 (Excitatory Amino Acid Transporter 2), expressed on the plasma membrane of pancreatic β-cells, is emerging as a crucial regulator of the islet glutamate clearance and β-cells survival. We here investigated whether alteration of EAAT2 may occur in type 2 diabetes mellitus (T2DM) and contribute to β-cell dysfunction. EAAT2 expression and localization were analysed by RNAseq and immunohistochemistry in human pancreases from healthy subjects and T2DM patients. Human islets exposed to metabolic insults and rodent β-cells were used to verify the molecular mechanisms involved. We observed an increased EAAT2 intracellular localization in islets of Langerhans from T2DM subjects as compared with healthy subjects, despite similar expression levels. Chronic treatment of islets from healthy donors with high glucose led to the transporter internalization in vesicular compartments and reduced [H3]-D-glutamate uptake, phenocopying the findings observed in T2DM sections. The transporter relocalization was associated to decreased P-Akt levels, suggesting an involvement of the PI3K/Akt pathway in the process. In accordance, PI3K inhibition by 100 µM LY294002 in human and clonal β-cells, caused the transporter internalization and significantly reduced the glutamate uptake compared to controls, suggesting that hyperglycemia changes the transporter trafficking to the plasma membrane. Upregulation of the glutamate transporter upon treatment with ceftriaxone rescued hyperglycemia-induced β-cells dysfunction and death by preventing the activation of NFκB pathway. Our data underscore the significance of EAAT2 in regulating islet physiology and provide a rationale for potential therapeutic targeting of this transporter to preserve β-cell survival and function in T2DM.