Adipose differentiation is regulated by several transcription factors, such as the CAAT/enhancer-binding protein family and peroxisome proliferator activator (PPAR) gamma2. Several recent studies have shown that the basic helix-loop-helix-PAS superfamily is also involved in the regulation of adipose differentiation. In this study, we investigated the roles played by EPAS1 (endothelial PAS domain protein 1) in adipogenesis. EPAS1, also referred to as hypoxia-inducible factor 2alpha, is a transcription factor known to play essential roles in catecholamine homeostasis, vascular remodeling, and the maintenance of reactive oxygen species, and so forth. During adipose differentiation in 3T3-L1 cells, the level of EPAS1 mRNA began to increase 6 days after the induction, and EPAS1 was highly expressed in differentiated cells. To examine whether EPAS1 is involved in adipogenesis, we first isolated stable clones from 3T3-L1 cells in which we could induce the expression of an EPAS1 C-terminal deletion mutant (designated EPAS1-(1-485)) with the insect hormone. The induction of EPAS1-(1-485) allowed the cells to accumulate only minimum amounts of intracellular lipid droplets. Consistent with the morphological observations, a minimum amount of aP2 and PPARgamma2 mRNA was induced in the EPAS1-(1-485) cells. We then examined whether or not EPAS1 was able to promote adipogenesis in NIH 3T3 cells, a relatively nonadipogenic cell line. Overexpression of EPAS1 in NIH 3T3 cells induced a significant amount of lipid accumulation compared with that of the control cells in the presence of the PPARgamma ligand. The results were also confirmed by measuring the expression of adipocyte-related genes. Adenovirus-mediated EPAS1-(1-485) expression resulted in the reduction of basal and insulin-dependent glucose transport in 3T3-L1 adipocytes. The mechanism involved the transcriptional regulation of GLUT1, GLUT4, and IRS3 expression by EPAS1. Taken together, these results suggest that EPAS1 plays several supporting roles in maintaining specific aspects of adipogenesis and adipocyte function including regulation of glucose uptake followed by lipid synthesis.