The mammalian target of rapamycin (mTOR) signaling network is frequently hyperactivated in patients with head and neck squamous cell carcinoma (HNSCC). Recent studies suggest that hypoxia, a common microenvironmental stress found in tumors, blocks this mitogenic pathway. Here, we demonstrate that in HNSCC cell lines, the expression of the phosphorylated forms of the mTOR downstream targets S6 kinase and S6 (pS6) decreased after hypoxia. These events were associated with a marked up-regulation of the regulated in development and DNA damage 1 (REDD1), a recently characterized hypoxia-induced protein that negatively controls mTOR activity. Conversely, pS6 levels were retained under hypoxia in REDD1 knock-down cells and in HNSCC cells lacking endogenous REDD1 expression. Furthermore, we observed that prolonged hypoxia induced an energy-depleting response as evidenced by decreased cellular ATP levels and AMP-activated protein kinase (AMPK) activation. Interestingly, AMPK inhibition before prolonged hypoxia prevented REDD1 expression, thereby sustaining mTOR activity. These results suggest a novel mechanism by which AMPK activation after hypoxia-induced energy stress may be crucial in regulating REDD1 expression to control the mTOR pathway in HNSCC. Furthermore, we found that, in some HNSCC cells, the reduced mTOR activity in response to hypoxia through AMPK/REDD1 was deregulated, which hence might contribute to the persistent activation of the mTOR pathway in this cancer type.