KRAS and BRAF mutations are frequently observed in human colon cancers. These mutations occur in a mutually exclusive manner, and each is associated with distinctive biological features. We showed previously that K-ras can interact with hypoxia to activate multiple signaling pathways. Many hypoxic responses are mediated by hypoxia-inducible factor (HIF)-1α and HIF-2α, and we sought to define the roles of mutant KRAS and BRAF in the induction of HIF-1α and HIF-2α in colon cancer cells. Ectopic expression of mutant K-ras in Caco2 cells enhanced the hypoxic induction of only HIF-1α, whereas mutant BRAF enhanced both HIF-1α and HIF-2α. Knockout or knockdown of mutant KRAS in DLD-1 and HCT116 cells impaired the hypoxic induction of only HIF-1α. HIF-1α mRNA levels were comparable in cells with and without a KRAS mutation. However, the rate of HIF-1α protein synthesis was higher in cells with a KRAS mutation, and this was suppressed by the phosphoinositide 3-kinase inhibitor LY294002. In contrast, knockdown of mutant BRAF in HT29 cells suppressed both HIF-1α and HIF-2α. Although BRAF regulated mRNA levels of both HIF-1α and HIF-2α, knockdown of BRAF or treatment with the MEK inhibitor PD98059 impaired the translation of only HIF-2α. Our data reveal that oncogenic KRAS and BRAF mutations differentially regulate the hypoxic induction of HIF-1α and HIF-2α in colon cancer, and this may potentially contribute to the phenotypic differences of KRAS and BRAF mutations in colon tumors. [Cancer Res 2009;69(21):8499–506]