Glucocorticoid-induced osteonecrosis of the femoral head (GIOFH) is a common and devastating orthopedic disease, and its underlying mechanism remains unclear. The aim of this study was to determine the role of microRNA-34a (mir-34a) in GIOFH. C57 mouse mesenchymal stem cells (mMSCs) and human umbilical vein endothelial cells (HUVECs) were cultured with dexamethasone (Dex). A total of 48 adult rats were treated with glucocorticoids, and after the onset of GIOFH, each femoral head was removed. Mir-34a mimics, an inhibitor and over-expressing lentivirus were used in vitro and in vivo, respectively. Real-time PCR, immunohistochemistry, ELISA, cell proliferation assays, osteoblastic differentiation, and endothelial activity assays were employed to evaluate the effect of mir-34a on mMSCs, osteoblasts, and vascular endothelial cells in glucocorticoid-treated mice. We found that Dex inhibited mMSC proliferation and osteoblastic differentiation, as well as the viability and activity of endothelial cells. Dex also caused osteonecrosis and decreased new vessel formation in vivo. Mir-34a alleviated the inhibitory effects of Dex on mMSCs and osteoblasts, while facilitating its inhibitory effects on endothelial cells. Mir-34a is an important regulator in osteogenesis and angiogenesis, and it might be useful as a therapeutic target for GIOFH. ? 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:417-424, 2018.