Here, we examined the role of the extracellular Ca(2+)-sensing receptor (CaSR) in the control of colonic epithelial cell proliferation in vivo and changes in β-catenin triggered by CaSR stimulation in human colonic epithelial cells in vitro. The in vivo studies, using a novel Casr intestinal-specific knock-out mouse, indicate that the genetic ablation of the Casr leads to hyperproliferation of colonic epithelial cells, expansion of the proliferative zone, changes in crypt structure, and enhanced β-catenin nuclear localization. The in vitro results indicate that stimulation of the CaSR, by Ca(2+) or by the calcimimetic R-568, produced a striking and time-dependent decrease in the phosphorylation of β-catenin at Ser-552 and Ser-675, two amino acid residues that promote β-catenin transcriptional activity. The reduced phosphorylation of β-catenin coincided with a decline in its nuclear localization and a marked redistribution to the plasma membrane. Furthermore, CaSR stimulation promoted a down-regulation of β-catenin-mediated transcriptional activation. These studies demonstrate that signaling pathways emanating from the CaSR control colonic epithelial cell proliferation in vivo and suggest that the mechanism involves regulation of β-catenin phosphorylation.