The ubiquitin-proteasome system is important to maintain pancreatic beta-cell function. Inhibition of the proteasome significantly reduced glucose-induced insulin secretion. Key regulators of the stimulus/secretion cascade seem to be affected by protein misfolding if the proteasome is down-regulated as recently reported in humans with Type 2 diabetes. It remains unknown, however, whether the glucose sensor enzyme glucokinase is involved in this process. A direct interaction between glucokinase and ubiquitin could be shown in vivo by FRET, suggesting regulation of glucokinase by the proteasome. After proteasome inhibition glucokinase activity was significantly reduced in MIN6 cells, whereas the protein content was increased, indicating protein misfolding. Enhancing the availability of chaperones by cyclohexamide could induce refolding and restored glucokinase activity. Glucokinase aggregation due to proteasome blocking with MG132, bortezomib, epoxomicin or lactacystin could be detected in MIN6 cells, primary beta-cells and hepatocytes using fluorescence-based assays. Glucokinase aggresome formation proceeded microtubule-assisted and was avoided by cyclohexamide. Thus the results of the present study provide support for glucokinase misfolding and aggregation in case of a diminished capacity of the ubiquitin-proteasome system in pancreatic beta-cells. In the Type 2 diabetic situation this could contribute to reduced glucose-induced insulin secretion.