The hypoxia-inducible factor-1 (HIF-1) transcription factor is an important regulator of tumor response to hypoxia that include increased angiogenesis, glycolytic metabolism, and resistance to apoptosis. HIF-1 activity is regulated by the availability of the HIF-1α subunit, the levels of which increase under hypoxic conditions. PX-478 (S-2-amino-3-[4′-N,N,-bis(2-chloroethyl)amino]phenyl propionic acid N-oxide dihydrochloride) is an inhibitor of constitutive and hypoxia-induced HIF-1α levels and thus HIF-1 activity. We report that PX-478 given to mice suppresses HIF-1α levels in HT-29 human colon cancer xenografts and inhibits the expression of HIF-1 target genes including vascular endothelial growth factor and the glucose transporter-1. PX-478 shows antitumor activity against established (0.15–0.40 cm³) human tumor xenografts with cures of SHP-77 small cell lung cancer and log cell kills up to 3.0 for other tumors including HT-29 colon, PC-3 prostate, DU-145 prostate, MCF-7 breast, Caki-1 renal, and Panc-1 pancreatic cancers. Large (0.83 cm³) PC-3 prostate tumors showed 64% regression, which was greater than for smaller tumors. The antitumor response to PX-478 was positively correlated with tumor HIF-1α levels (P < 0.02) and was accompanied by massive apoptosis. The results show that PX-478 is an inhibitor of HIF-1α and HIF-1 transcription factor activity in human tumor xenografts and has marked antitumor activity against even large tumor xenografts, which correlates positively with HIF-1α levels.