Silencing of GATA5 gene expression as a result of promoter hypermethylation has been observed in lung, gastrointestinal and ovarian cancers. However, the regulation of GATA5 gene expression has been poorly understood. In the present study, we have demonstrated that an E (enhancer)-box in the GATA5 promoter (bp -118 to -113 in mice; bp -164 to -159 in humans) positively regulates GATA5 transcription by binding USF1 (upstream stimulatory factor 1). Using site-directed mutagenesis, EMSA (electrophoretic mobility-shift analysis) and affinity chromatography, we found that USF1 specifically binds to the E-box sequence (5'-CACGTG-3'), but not to a mutated E-box. CpG methylation of this E-box significantly diminished its binding of transcription factors. Mutation of the E-box within a GATA5 promoter fragment significantly decreased promoter activity in a luciferase reporter assay. Chromatin immunoprecipitation identified that USF1 physiologically interacts with the GATA5 promoter E-box in mouse intestinal mucosa, which has the highest GATA5 gene expression in mouse. Co-transfection with a USF1 expression plasmid significantly increased GATA5 promoter-driven luciferase transcription. Furthermore, real-time and RT (reverse transcription)-PCR analyses confirmed that overexpression of USF1 activates endogenous GATA5 gene expression in human bronchial epithelial cells. The present study provides the first evidence that USF1 activates GATA5 gene expression through the E-box motif and suggests a potential mechanism (disruption of the E-box) by which GATA5 promoter methylation reduces GATA5 expression in cancer.