MicroRNAs (miRNAs) are small non-coding RNAs that repress protein production post-transcriptionally. MiRNAs play crucial roles in metabolism, endocrine cells development and in processes altered in T2D, such as insulin secretion. MiR-125b controls proliferation, apoptosis and differentiation of various cell types, although its role in β-cells remains unclear. Recent studies show an association between high levels of circulating miR-125b and hyperglycaemia (HbA1c) in prediabetes, T1D and T2D, suggesting this miRNA as a biomarker/contributor to the disease. I aim to determine whether glucose regulates miR-125b expression in β-cells and to understand its function by identifying its gene targets. We found that miR-125b expression is regulated by glucose in both mouse and human islets via AMP-activated protein kinase (AMPK), an important regulator of glucose homeostasis and target for anti-diabetic drugs. A combined analysis of both RNA-Seq and RNA-Immunoprecipitation of the miRNA-induced silencing complex (RIP-Seq) in β-cells overexpressing miR-125b identified dozens of novel miR-125b targets such as M6pr and Mtfp1, involved in enzyme sorting within secretory granules and mitochondrial fission and revealed a role for miR-125b in respiration and cytokine receptor interactions. Thus, miR-125b arises as an important regulator of β-cell function with a potential role in the deleterious effects of hyperglycaemia on β-cells.