The pro-inflammatory cytokines IL-1β and IFNγ play an important role in islet destruction leading to type 1 diabetes. 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) protects against insulitis and prolongs islet graft survival in NOD mice. Direct protection by 1,25(OH)2D3 in islets and decreased local inflammatory responses have previously been documented, but the mechanisms involved remain to be clarified. In this study we identified gene networks in islets involved in the protective actions of 1,25(OH)2D3 against pro-inflammatory cytokines. Human and mouse islets were exposed to IL-1β and IFNγ in the presence or absence of 10-8M 1,25(OH)2D3. Effects on insulin secretion and beta-cell survival were analyzed by glucose-stimulated insulin release and electron microscopy or Hoechst/PI staining, respectively. Gene expression profiles were assessed for mouse islets by Affymetrix microarrays. NF-κB activity was tested, while effects on secreted chemokines/cytokines were confirmed by ELISA and migration studies. Cytokine exposure caused a significant increase in beta-cell apoptosis in human and mouse islets, which was almost completely prevented by 1,25(OH)2D3. 1,25(OH)2D3 restored insulin secretion from cytokine-exposed human and mouse islets. Microarray analysis revealed that the expression of approximately 4000 genes was affected by cytokines (n=4, >1.3 fold, p<0.02), of which nearly 250 genes were modified by 1,25(OH)2D3. These genes belong to functional groups involved in immune response, chemotaxis, islet development, cell death and pancreatic beta-cell function/phenotype. Of interest, migration studies revealed that 1,25(OH)2D3 was able to inhibit the migration of monocytes towards cytokine-conditioned islet supernatant (n=7, p<0.05), confirming the functional relevance of decreased chemokine production/secretion when co-incubating cytokine-treated islets with 1,25(OH)2D3. In conclusion, these findings demonstrate a direct protective effect of 1,25(OH)2D3 against inflammation-induced beta-cell dysfunction and death in human and murine islets, with in particular alterations in chemokine production by the islets. These effects may contribute to the beneficial effects of 1,25(OH)2D3 against the induction of autoimmune diabetes.