CX3CL1/fractalkine is a chemokine with a unique CX3C motif. Hypoxia mediates the expression of various genes, such as vascular endothelial growth factor (VEGF), cyclooxygenase-2, and plasminogen-activator inhibitor-1, in vascular endothelial cells. We studied the effect of hypoxia on the expression of fractalkine induced by interferon-gamma (IFN-gamma) in endothelial cells. Human umbilical vein endothelial cells were cultured, and the stimulation of the cells with IFN-gamma was found to induce the expression of fractalkine. Hypoxia inhibited the expression of fractalkine mRNA and protein by IFN-gamma, and this effect was observed with concomitant increase in VEGF expression. Desferrioxamine, an iron chelator that mimics hypoxia in vitro, also inhibited the fractalkine production induced by IFN-gamma. Hypoxia did not affect the degradation of fractalkine mRNA. The inhibition of fractalkine expression by hypoxia was reversed on returning the cultures to reoxygenation condition. Inhibition of IFN-induced fractalkine expression by hypoxia was not affected by the presence of a radical scavenger, N-acetyl-L-cysteine, and the involvement of reactive oxygen species may be excluded. Inhibition of fractalkine expression by hypoxia may be involved in the pathophysiology of ischemic diseases.