Activation of PPARalpha has been demonstrated to inhibit tumor growth and angiogenesis, yet the mechanisms behind these actions remain to be characterized. The present study examined the effects of PPARalpha activation on the HIF-1alpha signaling pathway in human breast (MCF-7) and ovarian (A2780) cancer cells under hypoxia. Incubation of cancer cells under 1% oxygen for 16 hours significantly induced HIF-1alpha expression and activity as assayed with Western blot and reporter gene analysis. Treatment of the cells with PPARalpha agonists, but not a PPARgamma agonist, prior to hypoxia diminished hypoxia-induced HIF-1alpha expression and activity, and addition of a PPARalpha antagonist attenuated the suppression of the HIF-1alpha signaling. Activation of PPARalpha attenuated hypoxia-induced HA-tagged HIF-1alpha protein expression without affecting the HA-tagged HIF-1alpha mutant protein level, indicating that PPARalpha activation promotes HIF-1alpha degradation in these cells. This was further confirmed by the use of MG132, a proteasome inhibitor, which reversed PPAR£alpha-mediated suppression of the HIF-1alpha expression under hypoxia. Using the co-immunoprecipitation technique, we found that activation of PPARalpha enhances the binding of HIF-1alpha to Von Hippel-Lindau tumor suppressor (pVHL), a protein known to mediate HIF-1alpha degradation through the ubiquitin¡Vproteasome pathway. Following PPAalpha-mediated suppression of HIF-1alpha signaling, vascular endothelial growth factor (VEGF) secretion from the cancer cells was significantly reduced, and tube formation by endothelial cells was dramatically impaired. Taken together, these findings demonstrate for the first time that activation of PPARalpha suppresses hypoxia-induced HIF-1alpha signaling in cancer cells, providing novel insight into the anticancer properties of PPARalpha agonists.