Aberrant activation of the Wnt/beta-catenin signaling pathway is a hallmark of colon cancer. We show that the Wnt antagonist DICKKOPF-4 (DKK-4) gene is repressed by 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) in human colon cancer cells. This effect correlated with the inhibition of the DKK-4 promoter. Chromatin immunoprecipitation assays revealed that 1,25(OH)2D3 induces early and transient binding of the vitamin D receptor (VDR) and the SMRT corepressor to the region adjacent to the transcription start site of DKK-4. Additionally, we demonstrate that the DKK-4 gene is a new downstream target of TCF/beta-catenin. Remarkably, expression of DKK-4 messenger RNA (mRNA) was not detected in a series of 29 human normal (N) colon biopsies but expression was upregulated in all the matched tumour (T) tissues. An inverse correlation existed between the expression of DKK-4 and VDR RNA in the Ts. Ectopic DKK-4 expression increased the migration and invasion properties of colon cancer cells and conditioned media (CM) from DKK-4-expressing cells enhanced the capacity to migrate and form capillary-like tubules of human primary microvascular endothelial cells. In conclusion, DKK-4 is upregulated in colon cancer and is associated with the acquisition of malignant properties by neoplastic cells. DKK-4 downregulation is a novel effect of 1,25(OH)2D3 that may contribute to its anticancer action.