We have explored the optimal seeding density and timing for transplantation of the tissue-engineered bone with BMMSCs (bone marrow mesenchymal stem cells) and PDPB (partially deproteinized bone) in vitro. Rabbit BMMSCs of different densities were seeded into PDPB generated from fresh pig vertebrates to reconstruct tissue-engineered bone in vitro. Adhesion and proliferation of BMMSCs were analysed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay from which growth curves of BMMSCs on the PDPB materials were generated. The data show that BMMSCs began to adhere to PDPB after 24 h of primary culture, all groups reaching peak growth on the 6th day, after which the value of A decreased gradually and reached a plateau phase. The optimal BMMSCs seeding density of 5×106/ml achieved an excellent adhesion and proliferation activity on PDPB. In summary, the best cell seeding density of constructing tissue-engineered bone with BMMSCs in vitro is 5×106/ml, the optimal timing to transplant is the 6th day.