Type 1 diabetes is characterized by local inflammation (insulitis) in the pancreatic islets causing beta-cell loss. The mitochondrial p athway of apoptosis is regulated by the balance and interaction between Bcl-2 members. Here we clarify the molecular mechanism of beta-cell death triggered by the pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. The combination of TNF-alpha + IFN-gamma induced DP5, PUMA, and Bim expression in human islets and rodent beta-cells. DP5 and PUMA inactivation by RNA interference partially protected against TNF-alpha + IFN-gamma-induced beta-cell apoptosis. DP5 knockout mice had increased beta-cell area and isolated islets from these mice were resistant to cytokine exposure. Bim expression was transcriptionally regulated by STAT1 and its activation triggered cleavage of caspases. Silencing of Bim protected rodent and human beta-cells to a large extent against TNF-alpha + IFN-gamma, indicating a major role of this BH3-only activator protein in the mechanism of apoptosis. Our data support a highly regulated and context dependent modulation of specific Bcl-2 members controlling the mitochondrial pathway of beta-cell apoptosis during insulitis.