Vitamin D (VD) protects against colon carcinogenesis by mechanisms not fully understood. We had earlier reported on the similarity in the biologic action of VD and that of the calcium-sensing receptor (CaSR) in human colon carcinoma cells. At the molecular level, the CaSR gene contains 2 VD response elements and VD stimulates the expression of CaSR. In this study, we investigated on the relationship between VD action and CaSR function. We determined and compared the action of VD in human colon carcinoma cells (CBS, Moser, Caco-2 and HCT116) and their CaSR knocked-down counterparts. VD inhibited cellular proliferation, cellular invasion, and anchorage-independent growth and stimulated the expression of p21/Waf1 but not in CaSR knocked-down cells. These results demonstrate, for the first time, that the known tumor-suppressive function of VD requires functional CaSR and knocking down CaSR expression abrogated this function of VD. We recently reported that activation of CaSR in human colon carcinoma cells downregulated the expression of thymidylate synthase (TS) and survivin and promoted a significant increase in sensitivity to cytotoxic drugs. We now demonstrate, for the first time, that VD suppressed the expression of TS and survivin, TS and survivin gene transcriptional activities and promoted a cytotoxic response to 5-FU in a CaSR-dependent manner. Ectopic expression of wild-type CaSR in colon carcinoma cells also inhibited the expression of TS and survivin and enhanced cellular sensitivity to 5-FU. VD, however, could no longer enhance cellular sensitivity to 5-FU in cells overexpressing CaSR.