Inorganic polyphosphate [Poly(P)] induces differentiation of osteoblastic cells. In this study, matrix metalloproteinase (MMP)-3 small interfering RNA (siRNA) was transfected into purified rat dental pulp fibroblast-like cells (DPFCs) to investigate whether MMP-3 activity induced by Poly(P) is associated with cell differentiation into osteogenic cells. Real-time quantitative polymerase chain reaction, western blotting, and an MMP-3 activity assay were used in this study. Poly(P) enhanced expression of mature odontoblast markers dentin sialophosphoprotein (DSPP) and dentin matrix protein (DMP)-1 in DPFCs. These cells also developed an osteogenic phenotype with increased expression of osteocalcin (OC) and osteopontin (OP), high alkaline phosphatase (ALP) activity, and an increased calcification capacity. Poly(P) induced the expression of MMP-3 mRNA and protein, and increased MMP-3 activity. MMP-3 siRNA potently suppressed the expression of osteogenic biomarkers ALP, OC, OP, DSPP, and DMP-1, and blocked osteogenic calcification. Taken together, Poly(P)-induced MMP-3 regulates differentiation of osteogenic cells from DPFCs.