As part of its catabolic action in bone, parathyroid hormone (PTH) inhibits extracellular matrix mineralization. We previously showed that PTH dose-dependently induces matrix gla protein (MGP) expression in osteoblasts and this induction is at least partially responsible for PTH-mediated inhibition of mineralization. Recently, we identified PKA and ERK/MAPK as the key signaling pathways involved in PTH regulation of MGP expression. The goal of this study was to further characterize the mechanism by which PTH stimulates expression of MGP. Deletion analysis of the murine Mgp gene promoter identified a PTH-responsive region between −173 bp and−49 bp. Using gel-mobility shift assays we found that Sp1/Sp3, and Runx2 bind to distinct sites within this region. Mutation of either the Sp or the Runx2 site reduced MGP induction by PTH, while mutation of both sites completely abolished PTH responsiveness. Overexpression of Runx2 or Sp1 activated the Mgp reporter, while Sp3 was a dose-dependent repressor of Sp1 and PTH-induced MGP expression. Collectively, these data show that PTH regulates MGP gene transcription in osteoblasts through altered activities of Sp and Runx2 transcription factors. J. Cell. Biochem. 107: 284–292, 2009. © 2009 Wiley-Liss, Inc.