Objectives: To investigate the expression of microRNA-27a (miR-27a) in patients with steroid-associated femoral head osteonecrosis (SAN) and its regulatory effects on bone marrow mesenchymal stem cells (BMSCs). Methods: The expression levels of miR-27a and the osteogenic-related genes Runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 (BMP-2) were measured in bone marrow samples from SAN patients and controls. Human BMSCs were cultured in vitro and divided into four groups: blank control group, mimic negative control (mimic NC) group, miR-27a mimic group, and miR-27a inhibitor group. Cell proliferation, osteogenic differentiation, and expression of osteogenic-related genes were assessed. Results: The relative expression levels of miR-27a, Runx2, and BMP-2 in bone marrow tissues of the SAN group were significantly lower than those in the control group (all P<0.001). miR-27a expression was positively correlated with Runx2 and BMP-2 expression (P<0.001). Compared with the mimic NC group, the miR-27a mimic group showed increased cell proliferation (OD values), alkaline phosphatase (ALP) activity, number of calcium nodules, and expression levels of Runx2 and BMP-2 (all P<0.05). Compared with the mimic NC group, the miR-27a inhibitor group showed significantly decreased cell proliferation (OD values), ALP activity, number of calcium nodules, and expression levels of Runx2 and BMP-2 (all P<0.05). Conclusion: miR-27a may promote the proliferation and osteogenic differentiation of BMSCs by upregulating Runx2 and BMP-2 and may represent a potential therapeutic target for the treatment of SAN.