RCAN1 is a protein with known roles in the regulation of the protein phosphatase calcineurin, secretion in endocrine cells, mitochondrial function and has expression that is sensitive to Ca2+ and oxidative stress. As each of these is relevant to proper β-cell function or diabetes-associated β-cell dysfunction, we investigated whether RCAN1 has a functional role in β-cells. RCAN1 is expressed in MIN6 cells and mouse and human islets and gene expression is increased by high glucose and >20 fold in db/db islets. The overexpression of the human RCAN1.1 isoform in mice causes diabetes, age-associated hyperglycemia, decreased glucose tolerance, hypoinsulinemia, loss of β-cells, reduced insulin secretion, aberrant mitochondrial ROS production and the down regulation of key β-cell genes. In vitro patch clamp studies on single β-cells demonstrate an inability to undergo glucose-induced membrane depolarization in β-cells overexpressing RCAN1.1 despite any change in KATP channel functionality. We also observe exocytosis defects in these cells using membrane capacitance tracking, indicating direct effects of RCAN1 on the fusion of insulin granules with the plasma membrane. Our data therefore identifies a number of β-cell roles of RCAN1 and a potential link between hyperglycemia/diabetes and β-cell dysfunction.