Glucagon-like peptide-1 and its analogs may preserve pancreatic β-cell mass by promoting resistance to cytokine-mediated apoptosis. The mechanisms of TNFα-induced apoptosis and of its inhibition by exendin-4 were investigated in insulin-secreting cells. INS-1 and MIN6 insulinoma cells were exposed to 20 ng/ml TNFα, with or without pretreatment with 10 nm exendin-4. Treatment with TNFα increased c-Jun N-terminal protein kinase (JNK) phosphorylation 2-fold, reduced inhibitor-κBα (IκBα) protein content by 50%, induced opposite changes in caspase-3 and Bcl-2 protein content, and increased cellular apoptosis. Moreover, exposure to TNFα resulted in increased serine phosphorylation of both insulin receptor substrate (IRS)-1 and IRS-2 and reduced basal and insulin-induced Akt phosphorylation. However, in the presence of a JNK inhibitor, TNFα-induced apoptosis was diminished and serine phosphorylation of IRS proteins was prevented. When cells were pretreated with exendin-4, TNFα-induced JNK and IRS-1/2 serine phosphorylation was markedly reduced, Akt phosphorylation was increased, caspase-3 and Bcl-2 protein levels were restored to normal, and TNFα-induced apoptosis was inhibited by 50%. This was associated with a 2-fold increase in IRS-2 expression levels. A similar ability of exendin-4 to prevent TNFα-induced JNK phosphorylation was found in isolated pancreatic human islets. The inhibitory effect of exendin-4 on TNFα-induced JNK phosphorylation was abrogated in the presence of the protein kinase A inhibitor H89. In conclusion, JNK activation mediates TNFα-induced apoptosis and impairment of the IRS/Akt signaling pathway in insulin-secreting cells. By inhibiting JNK phosphorylation in a PKA-dependent manner, exendin-4 counteracts TNFα-mediated apoptosis and reverses the inhibitory events in the IRS/Akt pathway, resulting in promotion of cell survival.