Interleukin (IL)-2 knockout (KO) mice, which spontaneously develop symptoms of inflammatory bowel disease similar to ulcerative colitis in humans, were made vitamin D deficient (D-) or vitamin D sufficient (D+) or were supplemented with 1,25-dihydroxyvitamin D(3) (1,25D3). 1,25-Dihydroxyvitamin D3 supplementation, but not vitamin D supplementation, reduced the early mortality of IL-2 KO mice. However, colitis severity was not different in D-, D+, or 1,25D3 IL-2 KO mice. Cells from D- IL-2 KO mice produced more interferon (IFN)-gamma than cells from all other mice. Con A-induced proliferation was upregulated in IL-2 KO mice and downregulated in wildtype (WT) mice fed 1,25D3. All other measured immune responses in cells from IL-2 KO mice were unchanged by vitamin D status. In vitro addition of 1,25-dihydroxyvitamin D3 significantly reduced the production of IL-10 and IFN-gamma in cells from D- and D+ WT mice. Conversely, IFN-gamma and IL-10 production in cells from IL-2 KO mice were refractory to in vitro 1,25-dihydroxyvitamin D3 treatments. In the absence of IL-2, vitamin D was ineffective for suppressing colitis and ineffective for the in vitro downregulation of IL-10 or IFN-gamma production. One target of 1,25-dihydroxyvitamin D3 in the immune system is the IL-2 gene.