The present study investigated the role of mitogen-activated protein kinase (MAPK) signaling in osteoblastic differentiation of stromal ST-2 cells induced by synthetic octacalcium phosphate (OCP) incubation. Since our previous studies revealed that OCP consumes calcium ions in media during conversion to hydroxyapatite, the effect of the ions on ST-2 cell differentiation with or without OCP crystals was analyzed. The effect of presence or absence of MAPK inhibitors was also analyzed. OCP increased alkaline phosphatase (ALP) activity and the mRNA expression of differentiation markers via the p38 signaling pathway. The PD98059 MAPK inhibitor increased ALP activity and differentiation marker genes in cells cultured in OCP-coated wells. Reduction of calcium ions in the medium by EGTA increased the ALP activity without OCP in the presence of phosphate ion concentrations up to 7.5 mM. OCP may enhance osteoblastic differentiation through the p38 signaling pathway via the reduction of calcium ions induced by its physicochemical property.