Background: <p>Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder characterized by β-cell dysfunction and insulin resistance with limited treatment options. The targeting of pancreatic β-cell apoptosis and dedifferentiation represents a promising approach for T2DM therapy. Recently, mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have demonstrated repair potential for metabolic diseases. However, the effects of MSC-EVs on β-cell apoptosis and dedifferentiation in T2DM remain largely unclear.</p> Methods: <p>We established db/db mouse model and high glucose-stimulated MIN6 cell model to evaluate the therapeutic efficacy of MSC-EVs <i>in vivo</i> and <i>in vitro</i>. Transcriptome sequencing, proteomic analysis, and microRNA (miRNA) sequencing were applied to explore the molecular mechanism underlying MSC-EV-induced β-cell protection.</p> Results: <p>MSC-EVs significantly mitigated hyperglycemia, improved glucose tolerance, and restored β-cell function by preventing β-cell loss and reducing dedifferentiated β-cell populations in db/db mice. Mechanistically, MSC-EV treatment reversed hyperglycemia-induced ecotropic virus integration site 1 (EVI1) upregulation in β cells, and EVI1 knockdown significantly suppressed β-cell apoptosis and dedifferentiation by promoting forkhead box protein O1 (FOXO1) expression. MiRNA sequencing revealed that miR-4436 enriched in MSC-EVs inhibited EVI1 expression by directly binding to EVI1 mRNA. MiR-4436 knockdown abolished the therapeutic effects of MSC-EVs in T2DM</p> Conclusion: <p>MSC-EVs inhibit pancreatic β-cell loss and dedifferentiation by miR-4436-mediated EVI1/FOXO1 regulation. Therefore, MSC-EV administration may represent a promising therapeutic strategy for T2DM</p>