Inorganic phosphate (Pi) transport probably represents an important function of bone-forming cells in relation to extracellular matrix mineralization. In the present study, we investigated the effect of prostaglandin D2 (PGD2) on Pi transport activity and its intracellular signaling mechanism in MC3T3-E1 osteoblast-like cells. PGD2 stimulated Na-dependent Pi uptake time- and dose-dependently in MC3T3-E1 cells during their proliferative phase. A protein kinase C (PKC) inhibitor calphostin C partially suppressed the stimulatory effect of PGD2 on Pi uptake. The selective inhibitors of mitogen-activated protein (MAP) kinase pathways such as ERK, p38 and Jun kinases suppressed PGD2-induced Pi uptake. The inhibitors of phosphatidylinositol (PI) 3-kinase and S6 kinase reduced this effect of PGD2, while Akt kinase inhibitor did not. These results suggest that PGD2 stimulates Na-dependent Pi transport activity in the phase of proliferation of osteoblasts. The mechanisms responsible for this effect are activation of PKC, MAP kinases, PI 3-kinase and S6 kinase.