ackground Osteoprotegerin (OPG) and osteoprotegerin ligand (OPGL) play an important role in human bone metabolism. The aim of this research was to detect the expression of OPG and OPGL during human marrow stromal cells (hMSC) differentiation into osteoblasts (OB), and to observe their effect on osteoclasts (OC) formation in vitro to investigate bone metabolism mechanisms.Methods hMSCs were obtained from human bone marrow specimens using gradient centrifugation method, before being purified and incubated with differentiation medium to develop along the human osteoblasts (hOB) pathway. Morphology observation, biochemical detection and cell staining were performed during hMSC differentiation. OPG and OPGL mRNA levels were detected by reverse transcription-polymerase chain reaction. OPG and OPGL protein expression were determined by Western blotting. We further obtained OC progenitor cells from mice bone marrow and co-cultured with differentiating MSCs. We assessed the effect of OPG and OPGL on OC formation by identifying tartrate resistant acid phosphatase (TRAP) positive multinuclear cells.Results Optimal hMSC survival and purification were observed, along with stable biochemical indexes. Alkaline phosphatase secretion increased significantly and mineralization nodules appeared in the process of cell differentiation. OPG mRNA and protein level increased significantly, while OPGL mRNA and protein level decreased. Average levels of OPG mRNA and protein were about 2.5-fold higher than the control, while OPGL mRNA and protein levels were reduced by about one-half. In the group co-culturing with undifferentiated MSC or added OPGL, we found TRAP positive and multinuclear OC formation. However, OC formation was absent in the group co-culturing with differentiated MSC or added OPG.Conclusions During hMSC differentiation into hOB, OPG secretion increased rapidly and OPGL production decreased significantly. The OPG/OPGL ratio was also increased, while OC formation was inhibited and bone absorption decreased. Thus, regulation of the OPG/OPGL ratio may be important in controlling MSC differentiation, OB and OC formation in succession involved in bone metabolism.