Vitamin D is activated to its hormonal form, 1,25(OH)2D3 (calcitriol), by the CYP27B1 enzyme and conversely is inactivated by the CYP24A1 enzyme. This vitamin D metabolism centers on kidney regulation of CYP27B1 (induction by PTH, suppression by FGF23 and 1,25(OH)2D3), and reciprocal CYP24A1 suppression by PTH, and induction by FGF23 and 1,25(OH)2D3. This expression arises from complex and coordinated genomic regulation through unique enhancer modules. We discovered tissue-specific enhancers near Cyp27b1 in the mouse kidney that, when deleted, were coordinately responsible for PTH activation and FGF23 and 1,25(OH)2D3 suppression of this gene. Similarly, we found Cyp24a1 is regulated by a combination of distal and proximal enhancers for PTH, FGF23, and 1,25(OH)2D3. As expected, deletions of these enhancers had major impacts on calcium and phosphate homeostasis, and skeletal formation. Furthermore, these enhancer-deleted mice for both Cyp27b1 and Cyp24a1 have given us unique animals with specific disruption of vitamin D metabolism in only the kidney. Outside of the kidney, we are now able to leverage these mice to study inflammatory induction of 1,25(OH)2D3 production without systemic circulatory 1,25(OH)2D3 interference. Using on-tissue chemical derivatization and mass spectrometry imaging in our enhancer-deleted mice, we can, for the first time, quantify the levels of 1,25(OH)2D3 being made by non-renal tissues in vivo. In the absence of detectable 1,25(OH)2D3 in the kidneys, we found elevated levels of 1,25(OH)2D3 in the spleen and thymus in response to vitamin D supplementation. We also found suppression of cytokines like Tnfa and induction of Il4 in response to these high tissue 1,25(OH)2D3 levels. With an apparent non-renal 1,25(OH)2D3-induced modification of the inflammatory program, we are now investigating the physiologic impacts of vitamin D supplementation on amelioration of diseases such as atherosclerosis and inflammatory bowel disease (colitis) in a controlled and quantifiable way. Together, our studies outline the dynamic nature of vitamin D metabolism at the genomic level, assess non-renal response to supplementation, and begin to demonstrate the consequences of calcitriol production in health and in disease.