The aim of this study was to investigate whether an increase in the inorganic phosphate (Pi) concentration through the sodium-dependent phosphate cotransporters, Pit-1 and Pit-2 is involved in the differentiation of fibroblasts into osteoblasts in human PDLfs (hPDLfs). Expression of Pit-1 and Pit-2 mRNA from three different donors was examined by RT-PCR. Pit-1 and Pit-2 proteins were examined by western blotting. Localization of Pit-1 and Pit-2 was examined with immunocytochemistry. Phosphonoformic acid (PFA) was used to inhibit Pi transport. Time-course assays of Pi concentration in the hPDLfs were performed in the three different media; standard medium, calcification medium, and calcification medium with 1.0 mM PFA. Pit-1 and Pit-2 were expressed in hPDLfs at the mRNA and protein levels. The intracellular Pi (intra-Pi) concentration in hPDLfs incubated only in calcification medium was significantly higher than those in the other media, and calcification was observed in this medium. The intra-Pi concentration decreased by PFA, and calcification was not observed. The hPDLfs showed expression of Runx2 and osterix: specific marker of osteoblast in the calcification medium. The results indicated that the increase in the Pi concentration through Pit-1 and Pit-2 is play a critical role in the differentiation of PDLfs to osteoblasts.