Vitamin D/vitamin D receptor (VDR) signaling is reported to have a protective effect on the onset or progression of inflammatory bowel diseases (IBD), and hypoxia-inducible factor 1α (HIF-1α) activation is demonstrated to be closely associated with chemical-induced colitis. However, the association between vitamin D/VDR signaling and HIF-1α on IBD development remains a mystery. Here, we showed that HIF-1α expression was largely increased in the colonic epithelial cells of diseased tissues from patients with ulcerative colitis (UC). Consistently, HIF-1α activation was also improved in colonic epithelial cells upon TNFα treatment in a NF-κB pathway-dependent manner. HIF-1α inhibitors treatments ameliorated 2,4,6-trinitrobenzenesulfonic acid (TNBS)- or dextran sulfate sodium (DSS)-induced colitis in animal models. In cell or colitis animal models, vitamin D/VDR signaling suppressed HIF-1α overexpression in colonic epithelial cells via regulating NF-κB pathway, resulting in the inhibition of IFNγ and IL-1β overproductions in these cells. Collectively, these data suggest that vitamin D/VDR signaling relieves colitis development in animal models, at least in part, by suppressing HIF-1α expression in colonic epithelial cells. This study demonstrates vitamin D/VDR signaling inhibits colitis by suppressing HIF-1α activation in colonic epithelial cells. Since the effect of vitamin D/VDR signaling is only apparent on patients who seem to be vitamin D deficient, the benefits of vitamin D supplementation in patients who are not vitamin D deficient need to be proven.