Objective To investigate the role of miR-100-5p in the pathogenesis of non-traumatic osteonecrosis of the femoral head (NONFH). Methods The miRNA expression in patients with NONFH was detected by real-time quantitative PCR, the high expression of miR-100-5p in femoral head tissues of the patients determined. Rat bone marrow mesenchymal stem cells (rBMSCs) were cultured and divided into 5 groups: blank control group, dexamethasone treatment group (treated with dexamethasone for 3 days), miR-NC group (transfected with miR-NC), agomiR-100-5p group (overexpression of miR-100-5p), and antagomiR-100-5p group (transfected with miR-100-5p antagonist). The mRNA expression levels of miR-100-5p, alkaline phosphatase (ALP), Runt-associated transcription factor 2 (RUNX2), and collagen type I (Col1) were detected by real-time quantitative PCR. The protein expressions of ALP, RUNX2, Col1, and bone morphogenetic protein receptor 2 (BMPR2) were detected by Western blotting. The effect of miR-100-5p on the migration ability of rBMSCs was evaluated by scratch healing assay. And the effect of miR-100-5p on osteogenic differentiation ability of rBMSCs was investigated by alizarin red staining. Results miR-100-5p was significantly upregulated in the femoral head bone tissue of NONFH patients compared with normal femoral head bone tissue. Compared with those in the normal rBMSCs, the expression of miR-100-5p in rBMSCs treated with 20 μmol/L of dexamethasone was up-regulated. The upregulation of miR-100-5p in rBMSCs reduced the expressions of ALP, RUNX2, Col1, and BMPR2, and inhibited the osteogenic differentiation and migration abilities of rBMSCs. Conclusion The expression of miR-100-5p is elevated in bone tissues of NONFH patients and in rBMSCs treated with 20 μmol/L of dexamethasone. The up-regulated miR-100-5p may be involved in the pathogenesis of NONFH by inhibiting the migration and osteogenic differentiation of rBMSCs.