The interaction between HIF-1α, Mdm2, and p53 proteins during hypoxia has received recent attention. Here, we investigated the consequences of interaction between HIF-1α and Mdm2 under hypoxic conditions. Endogenous HIF-1α and Mdm2 proteins were co-immunoprecipitated from lysates of hypoxic HCT116 p53WT and p53−/− cells, suggesting that association of these two proteins is a p53-independent event. The cellular Mdm2 protein content was not significantly altered in hypoxic tumor cells. Overexpression of Mdm2 resulted in an increase in HIF-1α protein content in hypoxic cells and increased hypoxia-induced vascular endothelial growth factor (VEGF) transcriptional activation. These results point toward a novel and p53-independent function of Mdm2 to promote tumor cell adaptations to hypoxia by interacting with and promoting HIF-1 activation. © 2005 Wiley-Liss, Inc.