Patients with type 2 diabetes have lost the ability to maintain appropriate blood glucose levels due to a reduction of pancreatic beta cell mass and consequently of insulin release. A common observation in type 2 diabetes is an elevated urate plasma level or hyperuricemia, a substance that is known in the context of gout. Our aim was to identify the role of urate in pancreatic beta cell function and in the development of diabetes. Mouse (MIN6) and human (1.1B4) pancreatic beta cells were cultured under varying levels of urate over a range of different times and concentrations and in combination with normal or high glucose reflecting normal and diabetic conditions. Additionally, we have knocked-down GLUT9, a known urate transporter, via siRNAs in MIN6 cells to determine its contribution to the urate effects. The gout medication and urate transporter inhibitor benzbromarone was applied to test a clinical solution for the influence of hyperuricemia on beta cell function. Protein and total RNA was extracted and q-PCR and western blot analyses were performed to examine the levels of GLUT2, GLUT9, glucokinase, AMP-kinase and the miRNAs miR-153 and miR-34a. Moreover, we have performed caspase-3 apoptosis tests and MTT proliferation assays to evaluate the consequences of hyperuricemia on beta cell survival. In order to identify miR-153 as a regulator of GLUT9 we used a luc-GLUT9-UTR and a miR-153 overexpressing construct and employed luciferase assays. Diabetic conditions (high glucose and urate) resulted in no significant change of GLUT2 or glucokinase protein expression, which represent the glucose sensing machinery of pancreatic beta cells. However, expression of GLUT9 protein increased significantly especially under high glucose conditions. Expression of miR-34a, an important cell cycle regulator, changed in a time- and concentration-dependent manner upon exposure to hyperuricemic conditions. This could be attenuated by benzbromarone, a known uricosuric and GLUT9 inhibitor, and a knock-down of GLUT9, indicating that GLUT9 facilitates the urate effects in pancreatic beta cells. Hyperglycemic conditions significantly decreased expression of miR-153, which we identified as a regulator of GLUT9 expression, whereas hyperuricemic conditions revert this. Most interestingly, MIN6 and 1.1B4 cells exposed to hyperuricemic conditions showed an increase in AMPK/AMPK-P expression. Caspase-3 and MTT assays clearly reveal that hyperuricemia leads to apoptosis, which could be reverted by benzbromarone and GLUT9 knock-down.Our findings suggest an important impact of urate on beta cell function. Regulation of miR-153, which controls expression of its target gene GLUT9, may facilitate the urate-dependent rise in the pro-apoptotic miR-34a. More importantly, the significant increase in AMPK expression and activity induced by urate may ultimately cause inhibition of insulin secretion by inhibiting glycolysis, insulin synthesis and attenuating granular mobilization to the plasma membrane. In summary, we have identified a new mechanism and the involved transporter regulating insulin release and beta cell survival ultimately leading to the development of type 2 diabetes.