Introduction: MicroRNAs (MiRNAs) regulate multiple β-cell functions and represent mediators of islet impairment in type 2 diabetes (T2D). Human studies associating miRNAs with in vivo β-cell function are still lacking. The aim of this study is to investigate miRNAs expression in human islets derived from metabolically characterized living donors to identify pathways of β-cell dysfunction progression. Methods: 34 patients scheduled for partial pancreatectomy were subjected to OGTT and hyperglycemic clamp before surgery. Parameters of β-cell function were obtained, including β-cell Glucose Sensitivity (GS) and Rate Sensitivity (RS). RNA was extracted from Laser Capture microdissected islets and Small RNA Seq was performed. Differential Expression Analysis (DE)(Padj<0.05) and Linear Regression between miRNAs and clinical data were carried out. Validation of DE miRNAs was performed through ddPCR. Target genes were predicted using TargetScan7.2 and EndoC-βH1 cell line was transfected with miR-148a-3p. mRNA and protein levels of target genes were measured. Results: Subjects were stratified in 3 groups based on in-vivo measurements of GS/RS as index of progressive islet impairment: subjects with high GS and RS (functional; n=16), subjects with high GS and low RS (partially dysfunctional; n=10) and subjects with low GS and RS (severely dysfunctional; n=8). Six miRNAs resulted DE and miR-148a-3p was confirmed by ddPCR as increased during progressive β-cell dysfunction. Regression analysis showed that miR-148a-3p was negatively associated with GS and positively with mean glycaemia. TargetScan analysis revealed ATP6AP2, involved in GLP1R signalling, as a predicted target of miR-148a-3p. In EndoC-βH1 miR-148a-3p overexpression led to a decrease of ATP6AP2 mRNA (FC:0,43; p=0.03) and protein (FC:0,59;p=0,017). Conclusions: miR-148a-3p is associated with β-cell dysfunction and negatively regulates ATP6AP2, involved in fine-tuning of insulin secretion through GLP1R signalling. Our results suggest a new molecular axis to be explored as regulator of incretin-stimulated insulin secretion.