Insulin secreted from pancreatic β-cells and glucagon secreted from pancreatic α-cells are the two major hormones working in the pancreas in an opposing manner to regulate and maintain a normal glucose homeostasis. How microRNAs (miRNAs), a population of non-coding RNAs so far demonstrated to be differentially expressed in various types of cells, regulate gene expression in pancreatic β-cells and its closely associated α-cells is not completely clear. In this study, miRNA profiling was performed and compared between pancreatic β-cells and their partner α-cells. One novel miRNA, miR-483, was identified for its highly differential expression in pancreatic β-cells when compared to its expression in α-cells. Overexpression of miR-483 in β-cells increased insulin transcription and secretion by targeting SOCS3, a member of suppressor of cytokine signaling family. In contrast, overexpression of miR-483 decreased glucagon transcription and secretion in α-cells. Moreover, overexpressed miR-483 protected against proinflammatory cytokine-induced apoptosis in β-cells. This correlates with a higher expression level of miR-483 and the expanded β-cell mass observed in the islets of prediabetic db/db mice. Together, our data suggest that miR-483 has opposite effects in α- and β-cells by targeting SOCS3, and the imbalance of miR-483 and its targets may play a crucial role in diabetes pathogenesis.