This study was designed to evaluate the effect of miRNA acting in regulating multi-directional differentiation ability of mesenchymal stem cell in treatment of osteoporosis (OP), with the aim of finding a new idea and approach for clinical treatment of OP. Estrogen deficiency-induced OP mice model was established by means of ovariectomy (OVX). Additionally, a sham group was set up for control. Bone Marrow Mesenchymal Stem Cells (BMMSCs) of OVX group (O/BMMSCs) and BMMSCs of sham group (S/BMMSCs) were separately cultured. Then surface markers of BMMSCs were detected. Multi-directional differentiation ability was identified in the two groups by giving cells targeted induced stimulation. It was found that the bone trabecula, bone density and bone volume fraction of distal femoral metaphysis in the OVX group were much lower than those of the sham group. Moreover, trabecular bone space in the OVX group became larger; O/BMMSCs and S/BMMSCs both had normal expression of surface markers as well as potentials of osteogenic and adipogenic differentiation; O/BMMSCs had a weaker osteogenic capability but a stronger adipogenic capability than S/BMMSCs. All the findings suggest that the regulatory effect of miRNA on multi-directional differentiation ability plays a vital role in the treatment of OP, and there is a close correlation between them; deficiency or functional defect of BMMSCs can result in the occurrence of OP.