Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor composed of α and β subunits. HIF-1 is critically involved in cellular responses to hypoxia, glycolysis, and angiogenesis. Here, we show that treatment of prostate cancer PC-3 and LNCaP cells with the benzoquinone ansamycin geldanamycin, an Hsp90-specific inhibitor, induced degradation of HIF-1α protein in a dose- and time-dependent manner under both normoxia and hypoxia. This inhibition was also shown in other common cancer types tested. Rapid degradation of nuclear HIF-1α protein levels was accompanied by respective inhibition in HIF-1α functional transcription activity of VEGF. No difference between HIF-1α mRNA levels before or after geldanamycin treatment was found. Moreover, [35S]methionine pulse-chase analysis revealed that HIF-1α protein half-life was markedly decreased in the presence of geldanamycin compared with that in control. The geldanamycin-induced degradation of HIF-1α was reversed by proteosome inhibitors lactacystin and MG-132. We conclude that geldanamycin induces reduction of HIF-1α levels and its down-stream transcriptional activity by accelerating protein degradation independent of O2 tension. Thus, benzoquinone ansamycin drugs and their derivatives, such as 17-allyl-aminogeldanamycin, are excellent candidates as small molecule drug inhibitors of HIF-1 overexpression in cancer cells.