c-Myc (Myc) is a mediator of glucotoxicity but could also independently compromise β-cell survival and function. We have shown that after Myc activation in adult β-cells in vivo, apoptosis is preceded by hyperglycemia, suggesting glucotoxicity might contribute to Myc-induced apoptosis. To address this question conditional Myc was activated in β-cells of adult pIns-c-MycER(TAM) mice in vivo in the presence or absence of various glucose-lowering treatments, including exogenous insulin and prior to transplantation with wild-type islets. Changes in blood glucose levels were subsequently correlated with changes in β-cell mass and markers of function/differentiation. Activation of c-Myc resulted in reduced insulin secretion, hyperglycemia and loss of β-cell differentiation, followed by reduction in mass. Glucose-lowering interventions did not prevent loss of β-cells. Therefore, Myc can cause diabetes by direct effects on β-cell apoptosis even in the absence of potentially confounding secondary hyperglycemia. Moreover, as loss of β-cell differentiation/function and hyperglycemia are not prevented by preventing β-cell apoptosis, we conclude that Myc might contribute to the pathogenesis of diabetes by directly coupling cell cycle entry and β-cell failure through two distinct pathways.