To elucidate the potential function of microRNA-96 in protecting pancreatic beta cell function under the pathological condition of T2DM and the underlying mechanism. Relative levels of microRNA-96 and genes associated with beta cell function in the in vivo and in vitro T2DM and obesity models were detected by qRT-PCR. Insulin functions, including fasting blood glucose, plasma insulin, HOMA-IR, HOMA-%b, glucose tolerance and insulin tolerance, were assessed in microRNA-96 KO mice and wild-type mice fed with normal diet or high-fat diet. Downstream targets of microRNA-96 were verified by dual-luciferase reporter gene assay. Finally, regulatory effects of microRNA-96 on proliferation and apoptosis of MIN6 cells were determined. MicroRNA-96 was upregulated in mice fed with high-fat diet, db/db mice, highlevel glucose-treated cells, TNF-alpha-treated cells, pancreatic cells isolated from the obesity and T2DM patients. Increased fasting blood glucose and HOMA-IR, as well as decreased plasma insulin and HOMA-% b were observed in microRNA-96 KO mice. IPGTT and IPITT results indicated that knockout of microRNA96 led to pancreatic beta cell dysfunction under the pathological condition of T2DM. Dual-luciferase reporter gene assay confirmed that microRNA-96 could bind Foxo1 and Sox6. MicroRNA-96 negatively regulated Foxo and Sox6 levels. Moreover, overexpression of microRNA-96 promoted proliferative ability and inhibited apoptosis in MIN6 cells. Relative levels of Pdx1, Nkx6.1, Cyclin D1 and Cyclin E1 were upregulated in MIN6 cells overexpressing microRNA-96. Opposite results were obtained after knock-down of microRNA-96 in MIN6 cells. MicroRNA-96 is upregulated in pancreatic beta cells under the pathological condition of T2DM. Overexpression of microRNA-96 promotes proliferative ability and inhibits apoptosis in pancreatic beta cells through targeting Foxol and Sox6. (C) 2019 Elsevier Inc. All rights reserved.