Insulin secretion from pancreatic beta-cells occurs by sequential cellular processes, including glucose metabolism, electrical activity, Ca2+ entry, and regulated exocytosis. Abnormalities in any of these functions can impair insulin secretion. In the present study, we demonstrate that inhibition of proteasome activity severely reduces insulin secretion in the mouse pancreatic beta-cell line MIN6-m9. Although no significant effects on glucose metabolism including ATP production were found in the presence of proteasome inhibitors, both glucose- and KCl-induced Ca2+ entry were drastically reduced. As Ca2+-ionophore-induced insulin secretion was unaffected by proteasome inhibition, a defect in Ca2+ entry through voltage-dependent calcium channels (VDCCs) is the likely cause of the impaired insulin secretion. We found that the pore-forming alpha-subunit of VDCCs undergoes ubiquitination, which does not decrease but slightly increases expression of the alpha-subunit protein at the plasma membrane. However, electrophysiological analysis revealed that treatment with proteasome inhibitors results in a severe reduction in VDCC activity in MIN6-m9 cells, indicating that VDCC function is suppressed by proteasome inhibition. Furthermore, insulin secretion in isolated mouse pancreatic islets was also decreased by proteasome inhibition. These results demonstrate that the ubiquitin-proteasome system plays a critical role in insulin secretion by maintaining normal function of VDCCs.