Abstract: Objective: To investigate the effect of calcium hydroxide paste extract on the osteogenic differentiation of rat BMMSCs, and to provide theoretical basis for the clinical use of calcium hydroxide paste. Methods: Rat BMMSCs were isolated and cultured by whole bone marrow adherent method, and identified by flow cytometry. After induced by different conditions, Alizarin red staining, oil red O staining and alcian blue staining were used to identify the osteogenic, adipogenic and chondrogenic differentiation ability of stem cells. Calcium hydroxide paste was dried and ground into powder, then calcium hydroxide paste extract and calcium hydroxide paste conditioned medium (CH) were prepared. After being cultured with different concentrations of CH (0, 0.02, 0.2, 1, 1.5, 2 g/L)， ALP activity was measured to screen the best concentration for subsequent experiments. ALP staining, ALP activity assay and alizarin red staining were used to detect the changes in ALP activity and mineralization ability after CH induction. Western blot was used to detect the expression levels of COL1A1, ALP, RUNX2, and OSX. Results: Rat BMMSCs were successfully isolated and the cells had the characteristics of mesenchymal stem cells such as osteogenic, adipogenic and chondrogenic differentiation. 0.2 g/L was the optimal concentration of CH in inducing ALP activity of rat BMMSCs（P＜0.01）. The expressions of COL1A1, ALP, RUNX2 and OSX were up-regulated after the cells were treated with 0.2 g/L CH（P＜0.01）. Conclusion: Calcium hydroxide paste extract can potentially promote the osteogenic capacity of rat BMMSCs.