Tissue-specific expression of the human estrogen receptor alpha gene (ESR1) is achieved through multiple promoter sequences resulting in various mRNA transcripts encoding a common protein but differing in their 5'-untranslated region (5'-UTR). Many cancers are estrogen-sensitive with neoplastic growth stimulated through the estrogen receptor, a transcription factor that regulates developmental genes. We demonstrate that the human ESR1 gene is rich in potential quadruplex-forming sequences with 3 of 20 identified within exonic regions. In particular, we show using CD, UV, and NMR spectroscopy that a stable DNA G-quadruplex motif is formed within the exon C gene sequence. This motif, which PCR shows is transcribed in normal and neoplastic endometrium and in MCF-7 cells, forms a stable RNA quadruplex demonstrable by CD and UV analysis. Cloning the exon C G-quadruplex sequence upstream of a luciferase reporter gene caused a 6-fold reduction of enzymatic activity compared to a mutant sequence. We conclude that the exon C G-quadruplex motif is present in the 5'-UTR of the mRNA transcript, where it modulates the efficiency of translation.