Hypoxia-inducible factor 1 (HIF-1) plays a central role in cellular adaptation to changes in oxygen availability. Recently, prolyl hydroxylation was identified as a key regulatory event that targets the HIF-1alpha subunit for proteasomal degradation via the pVHL ubiquitination complex. In this report, we reveal an important function for ARD1 in mammalian cells as a protein acetyltransferase by direct binding to HIF-1alpha to regulate its stability. We present further evidence showing that ARD1-mediated acetylation enhances interaction of HIF-1alpha with pVHL and HIF-1alpha ubiquitination, suggesting that the acetylation of HIF-1alpha by ARD1 is critical to proteasomal degradation. Therefore, we have concluded that the role of ARD1 in the acetylation of HIF-1alpha provides a key regulatory mechanism underlying HIF-1alpha stability.