The aim of the present investigation was to characterize the role of the MAPK kinase kinase-1 (MEKK-1) in stress-induced cell death of insulin producing cells. We observed that transient overexpression of the wild type (wt) MEKK-1 protein in the insulin-producing cell lines RIN-5AH and TC-6 increased JNK phosphorylation and augmented cell death induced by DETA/NONOate (DETA/NO), Streptozotocin (STZ) and hydrogen peroxide (H₂O₂). Furthermore, DETA/NO or STZ induced a rapid threonine phosphorylation of MEKK-1. Silencing of MEKK-1 gene expression in TC-6 and human dispersed islet cells, using in vitro generated diced siRNA (d-siRNA), resulted in protection from DETA/NO, STZ, H₂O₂ and Tunicamycin induced cell death. Moreover, in DETA/NO-treated cells d-siRNA-mediated down-regulation of MEKK-1 resulted in decreased activation of JNK, but not of p38 and ERK. Inhibition of JNK by treatment with SP600126 partially protected against DETA/NO- or STZ-induced cell death. In summary, our results support an essential role for MEKK-1 in JNK-activation and stress-induced -cell death. Increased understanding of the signaling pathways that augment or diminish -cell MEKK-1 activity may aid in the generation of novel therapeutic strategies in the treatment of Type 1 diabetes.