Abstract: Objective：To investigate the role of the histone demethylases JMJD3 in regulating the pluripotency and the osteogenic differentiation of the bone marrow stromal cells（BMSCs）in vitro. Methods：Gradient centrifugation and adherent culture were used to isolate the BMSCs from rat tibia, and the lenti-virus of the JMJD3-shRNA and GFP (as the control) were constructed. The expression of H3K27me3 and the pluripotent transcription factors (Oct4/Nanog/Sox2) were detected by Western blot. The colony-forming efficiency was compared between JMJD3-shRNA group and GFP group. The different osteogenic ability of the JMJD3-shRNA group and the control group were analyzed by real-time RT-PCR, Western blot and Alizarin red staining after osteogenic induction. Results：Compared with the control, the expression of H3K27me3 was higher in JMJD3-shRNA group. The JMJD3 shRNA group possessed stronger colony-forming ability, expressed higher stemness markers (Oct4/Nanog/Sox2), compared with the control group. After 7-day osteogenic induction in vitro, the BMSCs’ osteogenic ability declined in JMJD3-shRNA group. After 14-day osteogenic induction, the calcium nodules of JMJD3-shRNA group were less than that of the control group. Conclusions：Inhibition of JMJD3 can enhance BMSCs’ stemness, and suppress their osteogenic differentiation.