Destruction of insulin-producing pancreatic β-cells by local autoimmune inflammation is a hallmark of type 1 diabetes. Histochemical analysis of pancreases from non-obese diabetic mice indicated activation of the transcription factor JunB/AP-1 (activator protein-1) after autoimmune infiltration of the islets. In vitro studies demonstrated that the cytokines tumor necrosis factor (TNF)-α and interferon (IFN)-γ induce JunB expression as a protective mechanism against apoptosis in both human and rodent β-cells. The gene network affected was studied by microarray analysis showing that JunB regulates nearly 20% of the cytokine-modified β-cell genes, including the transcription factor ATF3. Direct transcriptional induction of ATF3 by JunB is a key event for β-cell survival after TNF-α+IFN-γ treatment. Moreover, pharmacological upregulation of JunB/ATF3 via increased cAMP protected rodent primary β-cells and human islet cells against pro-inflammatory mediators. These results were confirmed in genetically modified islets derived from Ubi-JunB transgenic mice. Our findings identify ATF3 as a novel downstream target of JunB in the survival mechanism of β-cells under inflammatory stress.