Platelet-rich plasma (PRP), which contains growth factors for osteoblasts, is clinically used to promote regeneration of osseous tissue. However, we found that human plasma dose-dependently inhibits the growth of cultured MC3T3-E1 osteoblastic cells. In this study, the characteristics of osteoblast inhibiting factor in plasma were investigated. The inhibitory effect on MC3T3-E1 cells was inactivated by pre-heating the plasma. The molecular weight of this factor was estimated to be more than 100-kDaltons by the ultrafiltration method, and the macromolecular fraction strongly inhibited the spreading of MC3T3-E1 cells. Moreover, pre-treatment with a serine protease inhibitor abrogated the inhibitory effect of this factor, suggesting that serine proteases have an important role in inhibiting the growth of MC3T3-E1 cells. These results suggest that the improved preparation of platelets to exclude osteoblast-inhibiting factor from PRP is needed for the efficient regeneration of osseous tissue.