The basic molecular mechanism underlying mammalian oxygen-dependent regulation of hypoxia-inducible factor (HIF) via the von Hippel-Lindau E3 ubiquitin ligase is well established. The principal step in this critical cellular process is the hydroxylation of either or both of the two conserved proline residues P402 and P564 within the oxygen-dependent degradation domain (ODD) of HIF-1α subunit via prolyl hydroxylases, which is necessary for binding VHL. However, the significance of the two prolines has remained unclear considering that only one hydroxyproline is sufficient for the recruitment of VHL. Here, we show using biophysical analyses that both hydroxyprolines bind to the same interface on VHL with similar affinity; VHL binding affinity to HIF-1α ODD remains relatively unchanged regardless of whether the ODD contains one or two hydroxyprolines; ODD with two hydroxyprolines can accommodate two VHLs; and the rate of in vitro ubiquitination of ODD with one hydroxyproline via VHL E3 ligase is comparable to the rate observed with ODD containing two hydroxyprolines. However, the two hydroxyprolines show distinct contributions to the intracellular stability of HIF-1α ODD. These results demonstrate for the first time that the graduated HIF-1α stability profile observed over a range of oxygen tension is not attributed to the binding of or ubiquitination via VHL per se, but is likely due to the preceding events such as the efficacy of oxygen-dependent prolyl hydroxylase-mediated hydroxylation of HIF-1α.