Hypoxia-inducible factor (HIF)-1alpha undergoes degradation under normoxia, which involves its proline hydroxylation and subsequent binding of proline-hydroxylated HIF-1alpha to the von Hippel-Lindau protein-Elongin B-Elongin C (VBC) complex. In this study, we designed and synthesized a series of peptides containing 556-575 residues of HIF-1alpha with modifications at the Pro-564 residue to inhibit the interaction between proline-hydroxylated HIF-1alpha and VBC. Employing a fluorescence polarization-based interaction assay, we evaluated inhibitory potency of these peptides and selected potent inhibitors. We then analyzed their effects in the cell level to show that the selected inhibitors induced HIF-1alpha stabilization in normoxic cells. Considering that proline hydroxylation of HIF-1alpha is routinely targeted for modulating the HIF pathway, our approach of using inhibitors against the interactions between HIF-1alpha and VBC would provide an alternative way of upregulating HIF-1 activity.