An excessive and prolonged increase in glucose levels causes β-cell dysregulation, which is accompanied by impaired insulin synthesis and secretion, a condition known as glucotoxicity. Although it is known that both Lin28a and Lin28b regulate glucose metabolism, other molecular mechanisms that may protect against glucotoxicity are poorly understood. We investigated whether Lin28a overexpression can improve glucotoxicityinduced β-cell dysregulation in INS-1 and primary rat islet cells. INS-1, a rat insulinoma cell line was cultured and primary rat islet cells were isolated from SD-rats. To define the effect of Lin28a in chronic high glucose-induced β-cell dysregulation, we performed several in vitro and ex-vivo experiments. Chronic exposure to high glucose led to a downregulation of Lin28a mRNA and protein expression, followed by a decrease in insulin mRNA expression and secretion in β-cells. The mRNA and protein expression levels of PDX-1 and BETA2, were reduced; The levels of apoptotic factors, including c-caspase3 and the Bax/Bcl-2 ratio, were increased due to glucotoxicity. Adenovirusmediated Lin28a overexpression in β-cells reversed the glucotoxicity- induced reduction of insulin secretion and insulin mRNA expression via regulation of β-cell-enriched transcription factors such as PDX-1 and BETA2. Adenovirus-mediated overexpression of Lin28a downregulated the glucotoxicity-induced upregulation of c-caspase3 levels and the Bax/Bcl-2 ratio, while inhibition of endogenous Lin28a by small interfering RNA resulted in their up-regulation. Lin28a counteracted glucotoxicity-induced downregulation of p-Akt and p-mTOR. Our results suggest that Lin28a protects pancreatic β-cells from glucotoxicity through inhibition of apoptotic factors via the PI3 kinase/Akt/mTOR pathway. [BMB Reports 2021; 54(4): 215-220].