We studied the time courses of the number of osteoclasts, the expression of osteoblast differentiation markers and TGF-β1 levels in the platelet-poor plasma (PPP) in order to clarify the mechanism of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF)-induced bone formation in the mouse. Mice received PEG-rHuMGDF subcutaneously at a suprapharmacological dose (1.0 mg/kg) daily for 5 days (Day of the first injection: Day 1). PEG-rHuMGDF caused a gradual increase of platelet count, with a maximum increase on day 9. Histological analysis showed an increase of reticulin fibers on the inner side of endosteum on day 9, partial osteoid formation on day 11, new and excessive bone formation on day 13. On day 9, at the early stage of reticulin fiber increase, the number of osteoclasts was decreased. On day 11, osteopontin (OPN) mRNA-expression, a marker of osteoblast production, was observed in the reticulin fiber and osteoid near the substantia of bone marrow, whereas OPN mRNA-expressing cells did not express bone Gla protein (BGP) mRNA, a marker of osteoblasts, indicating that the expression of OPN mRNA was induced earlier than that of BGP mRNA during the course of osteogenesis. Additionally, the level of TGF-β1 in the PPP was increased, with about 2.5-fold elevation compared with the vehicle-treated mice at day 9. These findings suggest that PEG-rHuMGDF causes increase of reticulin fibers and osteogenesis by stimulating osteoblast differentiation and inhibiting osteoclast differentiation via an increase of the TGF-β1 level in the bone marrow.