Low circulating vitamin D levels have been linked to increased risks of cancer by epidemiologic studies, and preclinical research has demonstrated clear antitumor activities of vitamin D against various types of cancers. Calcitriol (1a, 25(OH)2 D3), the active form of vitamin D, has been shown to inhibit proliferation, induce differentiation, and activate apoptosis in colon cancer cells. However, the mechanism of calcitriol's antitumor action remains poorly defined and the hypercalcemic side effect of vitamin D limits its use as an anti-cancer agent. To understand its mechanism of action in tumor suppression, we investigated the effects of calcitriol on microRNA expression in colon cancer cells. We identified miR-627 as the microRNA whose expression was most significantly stimulated by calcitriol. Furthermore, JMJD1A (jumonji domain containing 1A), the gene encoding a histone demethylase, was identified as the target of miR-627. It has been reported that JMJD1A is upregulated in colon cancer during hypoxia. Increased JMJD1A expression decreases the histone methylation on the promoters of target genes such as adrenomedullin (ADM) and the growth and differentiation factor 15 (GDF15) and increases their expression, which promotes tumor growth. By downregulating JMJD1A, miR-627 mediated the effects of calcitriol to increase histone H3K9 methylation and to suppress the expression of growth-promoting genes, such as GDF15. Calcitriol also induced miR-627 and downregulated JMJD1A in colon cancer xenografts in nude mice. Calcitriol significantly suppressed the growth of colon tumors in nude mice, whereas colon tumor xenografts with stable JMJD1A-UTR expression showed resistance to the Calcitriol treatment, confirming the critical role of miR-627 in Calcitriol induced tumor suppression. Overexpression of miR-627 decreased JMJD1A and suppressed colon cancer growth both in vitro and in vivo. In addition, we found that miR-627 expression was decreased in human colon adenocarcinomas compared with controls. These results suggest that, miR-627 is a major epigenetic regulator in vitamin D induced growth inhibition. MiR-627 and JMJD1A may serve as potential targets to exploit the antitumor activity of vitamin D without eliciting its hypercalcemic side effect.