Objective  On the basis of preliminary evidence that microRNA(miR)-181b-5p inhibits osteogenic differentiation of human bone marrow mesenchymal stem cells (BMMSCs), the regulatory mechanism was further explored. Methods  Isolation, culture and identification of BMMSCs from the bone marrow of five healthy adults. The targeting relationship between miR-181b-5p and Sprouty 4(SPRY4) was investigated by bioinformatics software prediction, double luciferase reporter gene detection, Real-time PCR and Western blotting experiments. BMMSCs were divided into three groups, miR-181b-5p overexpression negative control group; miR-181b-5p overexpression group; miR-181b-5p overexpression +SPRY4 silenced group. Alkaline phosphatase(ALP) staining and ALP activity analysis were used to determine the effect of early osteogenic differentiation. The precipitation of calcium nodules was detected by alizarin red staining. The mRNA and protein expression levels of osteogenic differentiation marker genes were detected by Real-time PCR and Western blotting.  Results  BMMSCs were successfully isolated and identified. MiR-181b-5p specifically binds to the 3’UTR of SPRY4 mRNA. After overexpression of miR-181b-5p, the expression of SPRY4 protein level was significantly down-regulated, but there was no significant change in mRNA level. Knocking down the target gene SPRY4 blocked the effect of miR-181b-5p inhibitors on promoting osteogenic differentiation of cells. Conclusion  MiR-181b-5p inhibits osteogenic differentiation of BMMSCs by downregulating SPRY4 protein.