Abstract Tumor-initiating subpopulations of cancer cells, also known as cancer stem cells (CSC), were recently identified and characterized in prostate cancer. A well-characterized murine model of prostate cancer was used to investigate the regulation of hypoxia-inducible factor 1α (HIF1A/HIF1α) in CSCs and a basal stem cell subpopulation (Lin−/Sca-1+/CD49f+) was identified, in primary prostate tumors of mice, with elevated HIF1α expression. To further analyze the consequences of hypoxic upregulation on stem cell proliferation and HIF1α signaling, CSC subpopulations from murine TRAMP-C1 cells (Sca-1+/CD49f+) as well as from a human prostate cancer cell line (CD44+/CD49f+) were isolated and characterized. HIF1α levels and HIF target gene expression were elevated in hypoxic CSC-like cells, and upregulation of AKT occurred through a mechanism involving an mTOR/S6K/IRS-1 feedback loop. Interestingly, resistance of prostate CSCs to selective mTOR inhibitors was observed because of HIF1α upregulation. Thus, prostate CSCs show a hypoxic deactivation of a feedback inhibition of AKT signaling through IRS-1. In light of these results, we propose that deregulation of the PI3K/AKT/mTOR pathway through HIF1α is critical for CSC quiescence and maintenance by attenuating CSC metabolism and growth via mTOR and promoting survival by AKT signaling. We also propose that prostate CSCs can exhibit primary drug resistance to selective mTOR inhibitors. Implications: This work contributes to a deeper understanding of hypoxic regulatory mechanisms in CSCs and will help devise novel therapies against prostate cancer. Mol Cancer Res; 13(3); 556–64. ©2014 AACR.