Glucocorticoid-induced osteoporosis is a clinical problem in patients under chronic steroid therapy. To delineate the action of glucocorticoids on osteoblasts, we performed microarray analysis using rat primary osteoblasts and identified several glucocorticoid target genes. We validated the dexamethasone-induced upregulation of CCAAT/ enhancer-binding protein delta (C/EBPδ ), endothelial PAS-domain protein 1 (EPAS1), matrix Gla protein (MGP), and nerve growth factor inducible-B (NGFI-B) expression by quantitative real time-polymerase chain reaction (qPCR). EPAS1 overexpression inhibited, whereas dominant-negative EPAS1 overexpression enhanced the upregulation of osteoblastic marker genes and the mineralization in ST2 mesenchymal cells under the simulated osteoblastogenesis conditions. These results suggest that glucocorticoids could inhibit osteoblastic differentiation by regulating its target genes, as exemplified by EPAS1.