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NOX is the catalytic subunit of NADPH oxidase, the superoxide-generating enzyme. Among several isoforms of NOX, NOX4 is abundantly expressed in various tissues. To clarify the mechanisms of constitutive and ubiquitous expression of NOX4, the promoter activities of the human NOX4 gene were analyzed by reporter assays. The 5′-flanking and non-coding regions of the human NOX4 gene are known to contain multiple GC bases. Among them, three GC-boxes containing putative Sp/Klf-binding sites, which were not found in rodent genes, were suggested to be essential for the basal expression of the NOX4 gene in SH-SY5Y and HEK293 cells. Electrophoresis mobility shift assays demonstrated that Sp1 and Sp3 could bind to GC-boxes at positions −239/−227 and +69/+81 in these cells. Chromatin immunoprecipitation assays showed that Sp1 and Sp3 could also bind to GC-boxes at positions −239/−227 and +69/+81 in vivo. The promoter activity of the NOX4 gene was reduced in SH-SY5Y and HEK293 cells by transfection of an anti-Sp3 short hairpin RNA-expression plasmid. Taken together, these results suggest that Sp3 plays a key role in the expression of NOX4 in various cell lineages in humans.