The elucidation of the molecular mechanisms that govern the differentiation of bone marrow stromal cells (BMSCs) could provide new insight into the treatment of bone loss diseases. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression during stem cell growth, proliferation, and differentiation. In the present study, we showed that miR-338-3p expression was significantly down-regulated during the osteoblastic differentiation of BMSCs. Additionally, miR-338-3p was up-regulated in ovariectomized (OVX) mice compared with sham mice. An in vitro analysis revealed that the over-expression of miR-338-3p can inhibit the expression of osteoblast differentiation markers such as Osterix (Osx), thus reducing osteoblast differentiation. Bioinformatic analysis and dual luciferase assays confirmed that miR-338-3p can repress gene expression by targeting Runx2 and Fgfr2. In the BMSCs derived from OVX mice, the inhibition of miR-338-3p partially rescued mineralization and osteoblast differentiation. Taken together, our data show that miR-338-3p plays an important role during osteoblast differentiation of BMSCs and serves as a potential modulator of osteoporosis via its effect on osteoblasts.