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-1α 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-1α to regulate its stability. We present further evidence showing that ARD1-mediated acetylation enhances interaction of HIF-1α with pVHL and HIF-1α ubiquitination, suggesting that the acetylation of HIF-1α by ARD1 is critical to proteasomal degradation. Therefore, we have concluded that the role of ARD1 in the acetylation of HIF-1α provides a key regulatory mechanism underlying HIF-1α stability.