1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) is an immune modulator which prevents experimental autoimmune diseases. Receptors for 1,25-(OH)₂D₃ are present in pancreatic -cells, the target of an autoimmune assault in NOD mice. The aim of this study was to investigate the in vivo and in vitro effects of 1,25-(OH)₂D₃ on -cell gene expression and death and correlate these findings to in vivo diabetes development in NOD mice. When female NOD mice were treated with 1,25-(OH)₂D₃ (5 µg/kg/2d), there was a decrease in islet cytokine and chemokine expression, which was accompanied by less insulitis. Complementing these findings, we observed that exposure to 1,25-(OH)₂D₃ in three cell systems (INS-1^E cell line, FACS-purified rat -cells and NOD-SCID islets) suppressed IP-10 and IL-15 expression in the -cell itself but did not prevent cytokine-induced -cell death. This 1,25-(OH)₂D₃-induced inhibition of chemokine expression in -cells was associated with a decreased diabetes incidence in some treatment windows targeting early insulitis. Thus, while a short and early intervention with 1,25-(OH)₂D₃ (3 to 14 weeks of age) reduced diabetes incidence (35% vs. 58%, P 0.05), a late intervention (from 14 weeks of age, when insulitis is present) failed to prevent disease. Of note, only early and long-term treatment (3-28 weeks of age) prevented disease to a major extent (more than 30% decrease in diabetes incidence). We conclude that 1,25-(OH)₂D₃ mono-therapy is most effective in preventing diabetes in NOD mice when applied early. This beneficial effect of 1,25-(OH)₂D₃ is associated with decreased chemokine and cytokine expression by the pancreatic islets.