We have reported previously that PX-478 (S-2-amino-3-[4′-N,N,-bis(chloroethyl)amino]phenyl propionic acid N-oxide dihydrochloride) has potent antitumor activity against a variety of human tumor xenografts associated with the levels of the hypoxia-inducible factor-1α (HIF-1α) within the tumor. We now report that PX-478 inhibits HIF-1α protein levels and transactivation in a variety of cancer cell lines. Hypoxia-induced vascular endothelial growth factor formation was inhibited by PX-478, whereas baseline levels of vascular endothelial growth factor in normoxia were unaffected. Studies of the mechanism of PX-478 action showed that HIF-1α inhibition occurs in both normoxia and hypoxia and does not require pVHL or p53. In addition, PX-478 decreases levels of HIF-1α mRNA and inhibits translation as determined by ³⁵S labeling experiments and reporter assays using the 5′ untranslated region of HIF-1α. Moreover, to a lesser extent, PX-478 also inhibits HIF-1α deubiquitination resulting in increased levels of polyubiquitinated HIF-1α. The inhibitory effect of PX-478 on HIF-1α levels is primarily due to its inhibition of translation because HIF-1α translation continues in hypoxia when translation of most proteins is decreased. We conclude that PX-478 inhibits HIF-1α at multiple levels that together or individually may contribute to its antitumor activity against HIF-1α-expressing tumors. [Mol Cancer Ther 2008;7(1):90–100]