Adipose differentiation is regulated by coordination of several signaling pathways and transcription factors. We recently showed that Hypoxia inducible factor-2alpha (HIF-2alpha) plays several supporting roles in adipose differentiation and adipocytes functions including regulation of glucose uptake followed by lipid synthesis. HIF-2alpha expression is increased during adipogenesis, indicating that its up-regulation is necessary for execution of adipogenesis and maintenance of mature adipocytes functions. Therefore, in this study, to understand the mechanism by which HIF-2alpha expression is induced during adipogenesis, we investigated the promoter activity of HIF-2alpha gene during adipogenesis in 3T3-L1 cells. A comparison of HIF-2alpha promoter activity between preadipocytes and adipocytes revealed that the sequence -478/-445 is the putative core element that contributes to differentiation-dependent up-regulation of HIF-2alpha promoter activity. Electrophoretic mobility shift assays showed the presence of the specific nuclear factor bound to the sequence -478/-445 in both preadipocytes and adipocytes. Computer analysis revealed that this element contains several Sp1/Sp3 binding sites. Indeed, the presence of Sp1/Sp3 consensus oligonucleotides diminished the formation of the complexes composed of the sequence -478/-445 and the nuclear factor. Furthermore, specific retarded bands were supershifted with anti-Sp1 or -Sp3 antibodies. Binding of Sp1 and Sp3 to this element was also confirmed by chromatin immunoprecipitation analysis. The element encompassing -478/-445 favors Sp3 in preadipocytes and Sp1 in adipocytes. Finally, the activity of -478/-445 was increased by Sp1 but decreased by Sp3. Consequently, these results suggest that Sp1 and Sp3 are involved in transcriptional regulation of HIF-2alpha expression during adipogenesis in 3T3-L1 cells.