Objectives— Atherosclerotic lesions have regions that are hypoxic. Because the lesion contains macrophages that are loaded with lipid, we investigated whether hypoxia can influence the accumulation of lipids in these cells. Methods and Results— Exposure of human macrophages to hypoxia for 24 hours resulted in an increased formation of cytosolic lipid droplets and an increased accumulation of triglycerides. Exposure of the macrophages to oxidized low-density lipoprotein (oxLDL) increased the accumulation of cytosolic lipid droplets because of an increase in cellular cholesterol esters. The accumulation of lipid droplets in oxLDL-treated cells was further increased after hypoxia, caused by an increased level of triglycerides. Expression analyses combined with immunoblot or RT-PCR demonstrated that hypoxia increased the expression of several genes that could promote the accumulation of lipid droplets. Hypoxia increased the mRNA and protein levels of adipocyte differentiation-related protein (ADRP). It is well known that an increased expression of ADRP increases the formation of lipid droplets. Hypoxia decreased the expression of enzymes involved in β-oxidation (acyl-coenzyme A synthetase and acyl-coenzyme A dehydrogenase) and increased the expression of stearoyl–coenzyme A desaturase, an important enzyme in the fatty acid biosynthesis. Moreover, exposure to hypoxia decreased the rate of β-oxidation, whereas the accumulation of triglycerides increased. Conclusions— The results demonstrate that exposure of human macrophages to hypoxia causes an accumulation of triglyceride-containing cytosolic lipid droplets. This indicates that the hypoxia present in atherosclerotic lesions can contribute to the formation of the lipid-loaded macrophages that characterize the lesion and to the accumulation of triglycerides in such lesions.