AbstractMicroRNAs (miRNAs) regulate activities in living organisms through various signaling pathways and play important roles in the development and progression of osteoporosis. The balance between osteogenic and adipogenic differentiation of rBMSCs is closely related to the occurrence of osteoporosis. ERα regulates bone metabolism in various tissues. However, the correlation among ERα, miRNAs, and the differentiation of rBMSCs is still unclear. In this study, we used lentivirus transfection into rBMSCs to construct an ERα‐deficient model, analyzed the differences in expressed miRNAs between control and ERα‐deficient rBMSCs. The results revealed that the expression of 25 miRNAs were upregulated, 164 miRNAs were downregulated, and some of the regulated miRNAs such as miR‐210‐3p and miR‐214‐3p were related to osteogenic or adipogenic differentiation, as well as to particular signaling pathways. Next, we overexpressed miR‐210‐3p to evaluate its effects on the osteogenic and adipogenic differentiation of rBMSCs, and identified the relationship among miR‐210‐3p, Wnt signaling pathway, and the differentiation of rBMSCs. The results indicated that ERα‐deficient inhibited osteogenic differentiation, promoted adipogenic differentiation, and regulated the expression of some miRNAs. Meanwhile, overexpression of miR‐210‐3p promoted osteogenic differentiation and inhibited adipogenic differentiation of rBMSCs, processes likely to be related to the Wnt signaling pathway. In conclusion, we identified a group of upregulated and downregulated miRNAs in ERα‐deficient rBMSCs that might play a vital role in regulating osteogenic or adipogenic differentiation. One of these, miR‐210‐3p, inhibited osteogenic differentiation and promoted adipogenic differentiation correlated with the Wnt signaling pathway in ERα‐deficient rBMSCs, providing new insight into the regulation of bone metabolism.