We tested our novel hypothesis that down-regulation of hypoxia-inducible factor-1α (HIF-1α), the regulated subunit of HIF-1 transcription factor that controls gene expression involved in key functional properties of cancer cells (including metabolism, survival, proliferation, invasion, angiogenesis, and metastasis), contributes to a major antitumor mechanism of cetuximab, an approved therapeutic monoclonal antibody that blocks activation of the epidermal growth factor receptor. We showed that cetuximab treatment down-regulates HIF-1α levels by inhibiting synthesis of HIF-1α rather than by enhancing degradation of the protein. Inhibition of HIF-1α protein synthesis was dependent on effective inhibition of the phosphoinositide-3 kinase (PI3K)/Akt pathway by cetuximab, because the inhibition was prevented in cells transfected with a constitutively active PI3K or a constitutively active Akt but not in cells with a constitutively active MEK. Overexpression of HIF-1α conferred cellular resistance to cetuximab-induced apoptosis and inhibition of vascular endothelial growth factor production in sensitive cancer cell models, and expression knockdown of HIF-1α by RNA interference substantially restored cellular sensitivity to the cetuximab-mediated antitumor activities in experimental resistant cell models created by transfection of an oncogenic Ras gene (G12V) or by concurrent treatment of the cells with insulin-like growth factor-I. In summary, our data show that cetuximab decreases HIF-1α protein synthesis through inhibition of a PI3K-dependent pathway and that an effective down-regulation of HIF-1α is required for maximal therapeutic effects of cetuximab in cancer cells. [Mol Cancer Ther 2008;7(5):1207–17]