Expression of 25 hydroxyvitamin D 24 hydroxylase from the Cyp24a1 gene mediates 1,25 dihydroxyvitamin D (1,25(OH)2D) catabolism but gaps exist in our understanding of this enzyme's role in adult vitamin D (Vit D) metabolism and Vit D action in target tissues. To evaluate Cyp24a1 deletion in adult mice, we fed inducible-Cyp24a1 knockout (KO) heterozygous (HT), and control mice a chow diet from weaning and at 10 wks global recombination of floxed alleles was induced (50 mg Tamoxifen /g BW, ip, 5 d) and confirmed by PCR. At 11 wks, mice were fed an AIN93G diet for 1wk (0.5% Ca, 0.4% P, 200 IU/kg Vit D3) (n=10-14/group). In a second study, control (Cyp24a1flox/flox) and intestinal-epithelial-cell-specific knock-out mice (IEC KO, VillinCre+/- x Cyp24a1flox/flox, confirmed by PCR) were fed a chow diet from weaning until 11 wks then AIN93G diet for one week (n=12-15/group). At 12-wks mice were euthanized and serum was analyzed for Vit D metabolites by LC MS/MS while duodenal (Dd) and kidney (Kd) mRNA levels were quantified using qPCR. Contrary to reports showing lower serum 1,25(OH)2D in 60-d-old global Cyp24a1 KO mice (93 pM vs 264 pM in WT), in adult Cyp24a1 KO mice we saw higher levels of 25OHD (+185%) & 1,25(OH)2D (+41.4%) and reduced levels of 1,24,25(OH)3D (-53.7%). No changes in serum Vit D metabolites were seen in HT mice. The serum changes in adult KO mice were accompanied by a reduction in renal Cyp27b1 (-85.3%) and an increase in residual Cyp24a1 mRNA (+147%), demonstrating compensation for elevated 1,25(OH)2D levels. Kd Cyp27b1 mRNA was also reduced in HT mice (-36.5%), suggesting a milder, adaptive response. Despite higher serum 1,25(OH)2D in KO, no changes in Dd Trpv6 or S100g mRNA were observed and Cyp3a13 mRNA did not compensate for Cyp24a1 loss. Serum Vit D metabolites were not changed in IEC KO mice. However, there was a trend towards elevated renal Cyp24a1 mRNA (+61%, p=0.06), suggesting renal compensation to reduce renal 1,25(OH)2D production. Dd Trpv6 and S100g mRNA were not altered in IEC KO. Our data in adult KO mice indicate that Cyp24a1 has an important impact on physiologic Vit D regulation while our IEC KO data suggests that local degradation of the hormone by Cyp24a1 is not a strong regulator of intestinal Vit D action.