Hypoxia-inducible factor 1α (HIF-1α) is frequently overexpressed in human cancers and controls the expression of several genes that have been implicated in tumor growth and progression. Activity of HIF-1α in cancer cells is regulated at the transcriptional, translational and posttranslational level by multiple inter- and coacting molecular pathways. In this report, we reveal for the first time that tumor-associated CpG demethylation facilitates positive autoregulation of HIF-1α, resulting in amplification of hypoxia-induced transactivation of HIF-1α target genes. The HIF-1α promoter harbors a hypoxia response element that is normally repressed by methylation of a CpG dinucleotide located in the core element. In colon cancer cell lines and in primary colon cancer specimens, however, we found frequent aberrant demethylation of this element, enabling binding of HIF-1α to its own promoter resulting in autotransactivation of HIF-1α expression. Our results provide novel and highly unexpected insights into the complexity of HIF-1α regulation in cancer cells and implicate that tumor-associated CpG demethylation augments HIF-1α-mediated effects on malignant cell growth.