Although osteonecrosis of the femoral head is a known primary limitation of long-term or high-dose clinical administration of glucocorticoids, the mechanisms underlying this side effect remain unclear. Hypoxia is an important biological state under numerous pathological conditions. In this study, we investigated glucocorticoid-induced osteonecrosis under hypoxic conditions in the MC3T3-E1 osteoblast cell line using a cell cytotoxicity assay, flow cytometry, and western blotting. 6α-Methylprednisolone sodium succinate (MPSL) more effectively induced apoptosis and G0/G1 arrest of MC3T3-E1 osteoblasts under hypoxic conditions than under normoxic conditions. Correspondingly, MPSL more effectively upregulated cellular levels of cleaved caspase 3, p53, and its target p21, and downregulated cyclin D1 levels in hypoxia. Moreover, overexpression of Akt abrogated the MPSL activation of p53, p21, and cleaved caspase 3 and the attenuation of cyclin D1 expression and rescued osteoblasts from MPSL-induced cell cycle arrest and apoptosis, indicating that phosphatidylinositol 3-kinase (PI3K)/Akt signaling might play an essential role in MPSL-induced inhibition of osteoblasts. Furthermore, the suppression of PI3K/Akt signaling and upregualtion of cellular p85α monomer levels by MPSL were more pronounced under hypoxic conditions than under normoxic conditions. Finally, we found that the enhancement of the effects of MPSL under hypoxic conditions was attributed to hypoxia-upregulated glucocorticoid receptor activity. In conclusion, our results demonstrate that MPSL, a synthetic glucocorticoid receptor agonist, promotes the level of p85α and inhibits PI3K/Akt signaling to induce apoptosis and cell cycle arrest in osteoblasts, and that this effect is enhanced under hypoxic conditions.