Hypoxia inducible factor (HIF), a master regulator of critical genes for cell survival under hypoxic conditions, is known to be related to tumorigenesis and progression of renal cell carcinoma. N-methylpyrrole (Py)-N-methylimidazole (Im) hairpin polyamides are synthetic organic compounds that recognize and bind to the minor grooves of specific DNA sequences. We synthesized three Py-Im hairpin polyamides targeting the flanking sequences of hypoxia responsive element (HRE; a binding site of HIF) in the promoter region of the vascular endothelial growth factor (VEGF) gene. The effects of the polyamides on HIF-induced transcription were evaluated by a luciferase assay using a reporter plasmid containing a VEGF promoter. Real time reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay were performed to examine the effects of the polyamides on the transcription and secretion of VEGF in A498 renal cell carcinoma cells, which have a frame-shift mutation in the von Hippel-Lindau gene. A combination of three Py-Im hairpin polyamides suppressed HIF-induced transcription in reporter assays using 293 cells and successfully suppressed transcription and translation of the VEGF gene in A498 cells. Inhibition of the HIF-HRE interaction was confirmed by an electrophoresis mobility shift assay. An approach using Py-Im hairpin polyamides may be a new strategy for the treatment of renal cell carcinoma.