Low nutritional calcium contributes to disruption of the intestinal epithelial barrier function, to hyperproliferation of colonocytes and increased occurrence of aggressive secondary bile acids in the gut lumen. These mechanisms are also known to be involved in the etiology of colonic inflammation and cancer. We studied in mice and human adenocarcinoma-derived Caco-2 cells the impact of low calcium on markers of inflammation (cyclooxygenase-2; COX-2), of detoxification (pregnane and xenobiotic receptor (PXR)/steroid and xenobiotic receptor (SXR), cytochrome P450 steroid-inducible 3a11 (CYP3A11)), and on expression of the vitamin D system as a protection against tumorigenesis. Caco-2 cells express high COX-2 and low SXR mRNA levels when subconfluent. During differentiation this is reversed, while low calcium enhanced COX-2 protein expression. In vivo low dietary calcium significantly increased the expression of the PXR target gene CYP3A11 in the proximal colon, suggesting compensatory defense mechanisms. In comparison with males, low nutritional calcium elicits a better protective response in females: both the vitamin D synthesizing 25-hydroxyvitamin D3 1α hydroxylase (CYP27B1) mRNA and the detoxifying CYP3A11 mRNA are augmented more. While it is recognized that colonic vitamin D synthesis may prevent tumor progression, low dietary calcium also elevates the 1,25-(OH)2-D3 catabolic 25-hydroxyvitamin D3 24 hydroxylase (CYP24) expression primarily in the proximal colon. Our data suggest the proximal colon as the primary site of response to insufficient calcium intake.