MicroRNAs (miRNAs) are 19- to 25-nt fragments cleaved from 70- to 100-nt hairpin precursors. These molecules participate in essential biological processes. It was estimated that 30% of all protein-coding genes are miRNA targets. Thousands of miRNAs have already been identified in plants and animals, but little is known about their biological roles. MicroRNA375 (miR375) is highly expressed in pancreatic islets of humans and mice and regulates insulin secretion in isolated pancreatic cells. To improve our understanding of the biological roles of miR375, we constructed the plasmid pAAV-miR375 and transfected it into mouse Nit-1 cells. Real-time PCR and Northern blot analysis showed that the Nit-1 cells transfected with pAAV-miR375 over-expressed the mature miR375 compared with Nit-1 cells transfected with control plasmid or untransfected cells. The expression of myotrophin (Mtpn) decreased and insulin secretion was reduced in Nit-1 cells transfected with pAAV-miR375. In this study, we successfully established an over-expression system for miR375 and a technique to study the biological function of miRNAs by over-expression. We verified that miR375 reduced glucose-induced insulin secretion by down-regulating the expression of Mtpn in Nit-1 cells in vitro, suggesting that miR375 has potential therapeutic applications in type II diabetes.