Epidemiological data suggest a protective role of calcium and vitamin D against colorectal tumor pathogenesis. 1,25-dihydroxyvitamin D3 (1,25-D3) is a key determinant of calcium homeostasis, cell proliferation and differentiation. Calcium in the intestinal lumen functions as a growth regulator and may prevent cancer by direct reduction of colonocyte proliferation. While calcium or vitamin D can counteract proliferation by itself, they could also interact if nutritional calcium were to modulate colonic vitamin D synthesis. In this paper we demonstrate that colonic and renal vitamin D hydroxylases are regulated independently. When mice were fed a modified AIN-76 diet containing low dietary calcium (0.1 or 0.04%) fecal calcium content was as low as 5% of that found in mice on a 0.9% calcium containing diet. Low fecal calcium concentration enhanced proliferating cell nuclear antigen expression in the colon mucosa and reduced that of the cyclin dependent kinase inhibitor p21. While low dietary calcium did not affect colonic expression of VDR or 25-hydroxyvitamin D3 1alpha-hydroxylase (CYP27B1) mRNA, it influenced their renal expression in the expected manner by elevating the CYP27B1 expression and reducing VDR and 25-hydroxyvitamin D3 24-hydroxylase (CYP24) expression. In contrast, low calcium diets significantly augmented colonic CYP24 mRNA expression, but only in the ascending colon. This might result in reduced colonic accumulation of 1,25-D3 during hyperproliferation caused by low dietary calcium and might support site-specific tumorigenesis. The important realization that low dietary calcium by itself is a risk factor for colorectal carcinogenesis and that colonic and renal vitamin D hydroxylases indeed are regulated differently from each other will provide novel approaches for colon cancer prevention.