CD133 has been recognized as a specific cell surface marker for cancer stem cells in various tumors, although its biological functions and transcriptional regulation remain unclear. We found that the CD133 expression level was up-regulated in the lung cancer cell lines N417, H358, and A549, when these cell lines were cultured under hypoxic conditions. Among the five promoters (P1-P5) of human CD133 gene loci, P1 promoter was most strongly associated with hypoxia-induced promoter activity of CD133 gene expression. The P1 promoter possesses several cis-regulatory elements, including RUNT, GATA, ETS, OCT, SRY, and CREB-binding sites. A series of deletion and base substitution mutants of the P1 promoter revealed that OCT- and SRY-binding sites are important for hypoxia-induced promoter activity. The chromatin immunoprecipitation assay further confirmed the direct binding of Octamer biding trans-cription factor 3/4 (OCT4) and/or SRY-box containing gene 2 (SOX2) to the P1 promoter region of CD133 gene loci. In addition, the enhancement of both OCT4 and SOX2 expression by the α subunit of hypoxia-inducible factors (HIF1α and HIF2α) was required for hypoxia-induced CD133 expression. Knockdown of OCT4 or SOX2 expression in N417 cells with stabilized HIF1α and/or HIF2α abolished CD133P1 activity, while ectopic OCT4 or SOX2 expression triggers CD133P1 activity in the absence of HIF1α or HIF2α. Thus, in the hypoxic conditions, OCT4 and SOX2, both of which are induced by HIF1α/HIF2α. promote CD133 expression in the lung cancer cells via their direct interaction with the P1 promoter.