Abstract--Prolyl hydroxylase domain 2 protein (PHD2) signals the degradation of hypoxia-inducible factor (HIF)-1 by hydroxylating specific prolyl residues located within oxygen-dependent degradation domains. As expected, endothelial cells (ECs) overexpressing PHD2 had reduced HIF-1 and vascular endothelial growth factor-A expression and failed to accelerate their proliferation in response to hypoxia. Surprisingly, although these cells displayed further reductions in HIF-1 and vascular endothelial growth factor-A expression when cultured under normoxia, there was no further reduction in EC proliferation. Thus, there seemed to be no consistent correlation between PHD2 hydroxylase-mediated suppression of HIF-1 expression and inhibition of EC growth. Indeed, overexpression of a mutant PHD2 lacking hydroxylase activity also greatly diminished EC response to hypoxia-induced increase in proliferation, in spite of the fact that hypoxia-induced HIF-1 accumulation was not affected by mutant PHD2. These data strongly suggest the existence of a hydroxylase-independent mechanism for PHD2-mediated inhibition of EC proliferation under hypoxia. In support of a physiological relevance of PHD2 overexpression, we found that endogenous PHD2 expression was significantly upregulated by hypoxia and that silencing of the Phd2 gene by RNA interference significantly enhanced hypoxia-induced EC proliferation. In conclusion, this study demonstrates that PHD2 may act as a negative feedback regulator to antagonize hypoxia-induced EC proliferation.