Hypoxia-inducible factor-1alpha/beta (HIF-1alpha/beta) is a heterodimeric transcriptional activator that mediates gene expression in response to hypoxia. HIF-1alpha has been noted as an effective therapeutic target for ischemic diseases such as myocardiac infarction, stroke and cancer. By using a yeast two-hybrid system and a random peptide library, we found a 16-mer peptide named F29 that directly interacts with the bHLH-PAS domain of HIF-1alpha. We found that F29 facilitates the interaction of the HIF-1alpha/beta heterodimer with its target DNA sequence, hypoxia-responsive element (HRE). The transient transfection of an F29-expressing plasmid increases the expression of both an HRE-driven luciferase gene and the endogenous HIF-1 target gene, vascular endothelial growth factor (VEGF). Taken together, we conclude that F29 increases the DNA-binding ability of HIF-1alpha, leading to increased expression of its target gene VEGF. Our results suggest that F29 can be a lead compound that directly targets HIF-1alpha and increases its activity.