Objective: To investigate the expression of miRNA-34b and its effect and mechanism on osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). Methods: hBMSCs were isolated and cultured by the improved density gradient centrifugation combined with whole bone marrow adherent method, and cells were induced into osteoblasts by cultivation in osteogenic differentiation medium. The expression of miRNA-34b was detected by qRT-PCR and Western blot. Then miRNA-34b was overexpressed by transfection with miRNA-34b mimics and detected its influence on osteogenic differentiation of hBMSCs; meanwhile Western blot was used to analysis the activity of Notch pathway, in order to investigating its potential mechanism. Results: hBMSCs were successfully isolated and cultured in vitro and could be stably induced into osteoblasts when subjected to osteogenic differentiation medium. miR-34b expression decreased during the osteogenic differentiation of hBMSCs. Overexpression of miR-34b could suppress the ALP activity, mineral nodule formation and down regulate the osteogenic differentiation marker Runx2 (P<0.05). Furthermore, overexpression of miR-34b could suppress Notch pathway. Conclusion: miR-34b down regulates the osteogenic differentiation of hBMSCs and it is probably connected to the suppression of Notch pathway, which means that miR-34b could be a potential target for diagnosis and treatment of chronic inflammatory bone diseases.