Inorganic phosphate (Pi) and the matrix Gla protein (MGP) are key regulators of bone formation. We have recently shown that Pi upregulates MGP in growth plate chondrocytes, which may represent a negative feedback loop for the control of mineralization. Osteoblasts from Fra-1-deleted mice express low levels of MGP, whereas the expression of MGP is elevated in Fra-1 transgenic osteoblasts, suggesting a role for Fra-1 in MGP expression and bone formation. In this study, we aimed at deciphering the relationships between Pi and MGP in osteoblasts to determine the molecular mechanisms involved in the Pi-dependent regulation of MGP. In MC3T3-E1 cells and primary calvaria-derived osteoblasts, Pi increased MGP and Fra-1 expression at both the mRNA and protein levels. We also found that Pi enhanced the phosphorylation of ERK1/2. U0126 (MEK1/2 inhibitor) suppressed Pi-stimulated MGP and Fra-1 expression, indicating that ERK1/2 is required for Pi-dependent regulation of MGP and Fra-1. In addition, using in vitro DNA binding and chromatin immunoprecipitation assays, we showed that Fra-1 interacts with the MGP promoter in response to Pi in MC3T3-E1 cells. Finally, we found that in fra-1 knockdown MC3T3-E1 osteoblasts, the level of MGP expression is no more significantly upregulated by Pi. We further showed that primary osteoblasts from Fra-1-deficient mice failed to exhibit a Pi-dependent stimulation of MGP expression. These data show, for the first time, that Pi regulates MGP expression in osteoblasts through the ERK1/2-Fra-1 pathway.