Bone regeneration involves a series of events in a coordinated manner, including recruitment of mesenchymal stem cells, induction of immune response, inflammatory activity and vascular ingrowth. The microenvironment of bone regeneration is hypoxic. Low oxygen tension (hypoxia) promotes the upregulation of several signaling molecules. The primary mediating factor is the hypoxia-inducible factor-1 (HIF-1). Hypoxia stimulates the expression of a variety of cytokines from inflammatory cells, fibroblasts, endothelial cells, and osteoblasts. TNF-α is a key proinflammatory cytokine. The molecular events involved in osteoblast dysfunction under hypoxia are not fully understood. This study determined the effects of hypoxia on TNF-α in osteoblasts, and molecular mechanisms were explored. We observed that hypoxia induced TNF-α expression in a time-dependent manner in osteoblasts. Experiments using a potent HIF-1α activator DFO demonstrated that hypoxia-induced TNF-α was mediated by HIF-1-α. In addition, this study showed that hypoxia activated cyclooxygenase-2 (COX2) expression along with TNF-α. Inhibition experiments using COX2 inhibitor N398 indicated that COX2 was involved in hypoxia-mediated TNF-α expression, and this observation was further confirmed by Small interfering RNA against COX2. On the other hand, TNF-α didn't lead to the activation of COX2 expression. We conclude that COX2 is involved in hypoxia-induced TNF-α expression in osteoblast.