Epidemiological and preclinical data indicate that vitamin D and its most active metabolite 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] have anticancer activity. Accordingly, clinical trials are under way using several nonhypercalcemic 1alpha,25(OH)(2)D(3) analogues against various neoplasms including colon cancer. 1alpha,25(OH)(2)D(3) induces proliferation arrest and epithelial differentiation of human SW480-ADH colon cancer cells. We examined the gene expression profiles associated with 1alpha,25(OH)(2)D(3) exposure using oligonucleotide microarrays. 1alpha,25(OH)(2)D(3) changed the expression levels of numerous previously unreported genes, including many involved in transcription, cell adhesion, DNA synthesis, apoptosis, redox status, and intracellular signaling. Most genes were up-regulated, and only a small fraction were down-regulated. Fourteen of 17 candidate genes studied were validated as 1alpha,25(OH)(2)D(3) target genes by Northern and Western blotting or immunocytochemistry. They included c-JUN, JUNB, JUND, FREAC-1/FoxF1, ZNF-44/KOX7, plectin, filamin, keratin-13, G(0)S2, and the putative tumor suppressors NES-1 and protease M. There was little overlap between genes regulated after short (4 h) or long (48 h) exposure. Gene regulatory effects of 1alpha,25(OH)(2)D(3) in SW480-ADH cells differed from those in LS-174T cells, which lack E-cadherin and do not differentiate in response to 1alpha,25(OH)(2)D(3). Data from this study reveal that 1alpha,25(OH)(2)D(3) causes a profound change in gene expression profiles and provide a mechanistic basis to the ongoing clinical studies using nonhypercalcemic vitamin D(3) derivatives for colon cancer prevention and treatment.