The active form of vitamin D [1,25-dihydroxycholecalciferol, 1,25(OH)2D3] and the vitamin D receptor (VDR) regulate susceptibility to experimental colitis. The effect of the bacterial microflora on the susceptibility of C57BL/6 mice to dextran sodium sulfate-induced colitis was determined. Mice that cannot produce 1,25(OH)2D3 [Cyp27b1 (Cyp) knockout (KO)], VDR KO as well as their wild-type littermates were used. Cyp KO and VDR KO mice had more bacteria from the Bacteroidetes and Proteobacteria phyla and fewer bacteria from the Firmicutes and Deferribacteres phyla in the feces compared with wild-type. In particular, there were more beneficial bacteria, including the Lactobacillaceae and Lachnospiraceae families, in feces from Cyp KO and VDR KO mice than in feces from wild-type. Helicobacteraceae family member numbers were elevated in Cyp KO compared with wild-type mice. Depletion of the gut bacterial flora using antibiotics protected mice from colitis. 1,25(OH)2D3 treatment (1.25 μg/100 g diet) of Cyp KO mice decreased colitis severity and reduced the numbers of Helicobacteraceae in the feces compared with the numbers in the feces of untreated Cyp KO mice. The mechanisms by which the dysbiosis occurs in VDR KO and Cyp KO mice included lower expression of E-cadherin on gut epithelial and immune cells and fewer tolerogenic dendritic cells that resulted in more gut inflammation in VDR and Cyp KO mice compared with wild-type mice. Increased host inflammation has been shown to provide pathogens with substrates to out-compete more beneficial bacterial species. Our data demonstrate that vitamin D regulates the gut microbiome and that 1,25(OH)2D3 or VDR deficiency results in dysbiosis, leading to greater susceptibility to injury in the gut.