Fetoplacental endothelial cells are exposed to oxygen levels ranging from 2% to 8% in vivo. However, little is known regarding endothelial function within this range of oxygen because most laboratories use ambient air (21% O₂) as a standard culture condition (SCN). We asked whether human umbilical artery endothelial cells (HUAECs) that were steadily exposed to the physiological chronic normoxia (PCN, 3% O₂) for ∼20–25 days differed in their proliferative and migratory responses to FGF2 and VEGFA as well as in their global gene expression compared with those in the SCN. We observed that PCN enhanced FGF2- and VEGFA-stimulated cell proliferation and migration. In oxygen reversal experiments (i.e., when PCN cells were exposed to SCN for 24 h and vice versa), we found that preexposure to 21% O₂ decreased the migratory ability, but not the proliferative ability, of the PCN-HUAECs in response to FGF2 and VEGFA. These PCN-enhanced cellular responses were associated with increased protein levels of HIF1A and NOS3, but not FGFR1, VEGFR1, and VEGFR2. Microarray analysis demonstrated that PCN up-regulated 74 genes and down-regulated 86, 14 of which were directly regulated by hypoxia-inducible factors as evaluated using in silico analysis. Gene function analysis further indicated that the PCN-regulated genes were highly related to cell proliferation and migration, consistent with the results from our functional assays. Given that PCN significantly alters cellular responses to FGF2 and VEGFA as well as transcription in HUAECs, it is likely that we may need to reexamine the current cellular and molecular mechanisms controlling fetoplacental endothelial functions, which were largely derived from endothelial models established under ambient O₂.