We have previously shown that the extracellular calcium-sensing receptor (CaR) is expressed in various bone marrow-derived cell lines and plays an important role in stimulating their proliferation and chemotaxis. It has also been reported that the CaR modulates matrix production and mineralization in chondrogenic cells. However, it remains unclear whether or not the CaR plays any role in regulating osteoblast differentiation. In this study, we found that the mineralization of the mouse osteoblastic MC3T3-E1 cells was increased when the cells were exposed to high calcium (2.8 and 3.8 mM) or a specific CaR activator, NPS R467 (1 and 3 µM). Next, we stably transfected MC3T3-E1 cells with either a CaR antisense vector (AS clone) or a vector containing the inactivating R185Q-variant of the CaR (DN clone) that has previously been shown to exert a dominant negative action. The ALP activities were decreased compared with controls in both the AS and DN clones. However, the levels of type I procollagen (COLI) and osteopontin (OPN) mRNA in the AS clone, as detected by Northern blotting, were almost the same as in the controls. On the other hand, the expression of osteocalcin (OCN), which is expressed at a later stage of osteoblastic differentiation, was significantly reduced in both the AS and DN clones. Mineralization was also decreased in both clones. In conclusion, this study showed that the abolition of CaR function results in diminishing ALP activity, OCN expression and mineralization in mouse osteoblastic cells. This suggests that the CaR may be involved in osteoblastic differentiation.