Four putative G-quadruplex sequences (PGSs) in the HIF1 alpha promoter and the 5'UTR were evaluated for their G-quadruplex-forming potential using ESI-MS, CD, FRET, DMS footprinting, and a polymerase stop assay. An important G-quadruplex (S1) has been proven to inhibit HIF1 alpha transcription by. blocking AP2 binding. A benzo[c]phenanthridine derivative was found to target the S1 G-quadruplex and induce its conformational conversion from antiparallel to parallel orientation. The transcriptional suppression of HIF1 alpha by this compound was demonstrated using western blotting, Q-RT-PCR, luciferase assay, and ChIP. Our new findings provided a novel strategy for HIF1 alpha regulation and potential insight for cancer therapy.