Malignant insulinoma is a critical cancer form with a poor prognosis. Since cure by surgery is infrequent, effective chemotherapy is in demand. Induction of cell death in tumor cells by proteasome inhibitors is emerging as a potential strategy in cancer therapy. Here we investigated whether inhibition of the proteasome has an anti-tumorigenic potential in insulinoma cells. Exposure of mouse TC3 insulinoma cells to the proteasome inhibitor N-Acetyl-Leu-Leu-Nle-CHO (ALLN) reduced cell viability, activated caspase-3, induced apoptosis and suppressed insulin release. Treatment with ALLN also resulted in phosphorylation of c-jun N terminal kinase (JNK) and an increase in in vitro phosphorylation of c-jun. In insulinoma cells with impaired JNK signaling, ALLN-induced apoptosis was significantly suppressed. Another proteasome inhibitor, lactacystin (LC), also stimulated JNK activation, caused activation of caspase-3, suppressed cell viability and induced apoptosis in TC3 and rat INS-1E cells. Both ALLN and LC caused a marked decrease in the cellular amount of the JNK scaffold protein JIP-1/IB1. In primary pancreatic rat islet cells, proteasome inhibition reduced insulin secretion, but had no impact on cell viability and even partially protected against the toxic effect of pro-inflammatory cytokines. Our findings demonstrate that proteasome inhibitors possess anti-tumorigenic and anti-insulinogenic effects on insulinoma cells.