Tissue hypoxia is a common feature in solid tumors. Hypoxia-inducible factor 1 (HIF-1) is a critical transcription factor that regulates the expression of genes encoding factors that influence tumor growth including vascular endothelial growth factor. Previous studies have demonstrated that post-transcriptional modification events are important for regulation of HIF-1alpha protein expression and HIF-1 transcriptional activity. Prostaglandin E2 (PGE2), a major end product of the cyclooxygenase-2 (COX-2) enzyme, induces the basal and hypoxia-induced nuclear relocalization of HIF-1alpha. This is suppressed by NS398, a COX-2 selective inhibitor. NS398 also inhibits hypoxia-induced angiogenesis, which may be mediated by the inhibition of HIF-1 function in a COX-2-dependent manner. Here, we show that NS398 reduces HIF-1alpha and HIF-1 transcriptional function in both COX-2 positive PC-3 cells and COX-2 negative HCT116 cells under normoxic and hypoxic conditions. On the one hand, NS398 decreases the expression of HIF-1alpha mRNA and reduces HIF-1alpha synthesis in a COX-2/PGE2 dependent way, which can be restored by addition of exogenous PGE2 that activates the phosphatidylinositol 3-kinase/AKT/p70s6k signaling pathway. On the other hand, NS398 accelerates HIF-1alpha degradation by moderately increasing ubiquitination and remarkably promoting the clearance of ubiquitylated protein, an effect most likely independent of COX-2/PGE2 since exogenous PGE2 fails to reverse it. Finally, NS398 decreases hypoxia-induced shifted form of HIF-1alpha and attenuates HIF-1 activation in greater extent under hypoxic than normoxic conditions. These data not only confirm the inhibitory effect of NS398 on HIF-1alpha and HIF-1 transcriptional activity but also demonstrate that such an effect occurs at multiple levels involving both COX-2 dependent and independent mechanisms.