▸ We investigate the effects of 1,25(OH)2D3 on global gene expression in pancreatic islets. ▸ Cytoskeletal and intracellular trafficking genes are induced by 1,25(OH)2D3 in islets. ▸ Genes involved in intercellular junctions in islets are upregulated by 1,25(OH)2D3. ▸ 1,25(OH)2D3 affects expression of genes associated with lipid metabolism in islets. ▸ Genes involved in ion transport and homeostasis in islets are induced by 1,25(OH)2D3. Vitamin D deficiency has been linked to type 1 and 2 diabetes, whereas supplementation may prevent both diseases. However, the extent of the effects of vitamin D or its metabolites directly on pancreatic islets is still largely unknown. The aim of the present study was to investigate how active vitamin D, 1,25(OH)2D3, affects beta cells directly by establishing its effects on global gene expression in healthy murine islets. Pancreatic islets were isolated from 2 to 3 week old C57BL/6 mice and cultured in vitro with 1,25(OH)2D3 or vehicle for 6 and 24h. Total RNA was extracted from the islets and the effects on global gene expression were analyzed using Affymetrix microarrays. Exposure to 1,25(OH)2D3 compared to vehicle resulted in 306 and 151 differentially expressed genes after 6 and 24h, respectively (n=4, >1.3-fold, p<0.02). Of these 220 were up-regulated, whereas 86 displayed a decreased expression after 6h. Furthermore, expression levels were increased for 124 and decreased for 27 genes following 24h of exposure. Formation of intercellular junctions, cytoskeletal organization, and intracellular trafficking as well as lipid metabolism and ion transport were among the most affected gene classes. Effects on several genes already identified as being part of vitamin D signaling in other cell types were observed along with genes known to affect insulin release, although with our assay we were not able to detect any effects of 1,25(OH)2D3 on glucose-stimulated insulin release from healthy pancreatic islets. The effects of 1,25(OH)2D3 on the expression of cytoskeletal and intracellular trafficking genes along with genes involved in ion transport may influence insulin exocytosis. However, an effect of 1,25(OH)2D3 on insulin release could not be detected for healthy islets in contrast to islets subjected to pathological conditions such as cytokine exposure and vitamin D deficiency as suggested by other studies. Thus, in addition to previously identified tolerogenic effects on the immune system, 1,25(OH)2D3 may affect basic functions of pancreatic beta cells, with the potential to render them more resistant to the detrimental conditions encountered during type 1 and 2 diabetes. This article is part of a Special Issue entitled ‘Vitamin D Workshop’.