Among Tissue kallikrein genes (KLKs), KLK1 is abundantly expressed in human skin. Although its putative promoter is known to have various cis-elements, they have not been functionally tested. In the present study, the regulation mechanism of KLK1 promoter supporting such abundant expression was examined. Luciferase assay targeting the KLK1 promoter (nucleotide −1153/+40 from the major transcriptional start site) was performed on NHEK human keratinocyte. −954/−855, −428/−236, and −100/+40 had the induction activity. The motif search program failed to find unique binding motifs in −428/−236, whereas both −954/−855 and −100/+40 had a unique GATAs binding motif. Electrophoretic mobility shift assay (EMSA) and DNA footprinting confirmed the binding of NHEK nuclear protein to these motifs that were supershifted by anti-GATA3 antibody. Among GATA isoforms, GATA3 alone could be amplified in RNA obtained from NHEK. Moreover, introduction of GATA3 into fibroblastic NIH3T3 cells enhanced the activity of KLK1 promoter containing −954/+40, while that of GATA3 dominant negative mutant to NHEK cells impaired the same promoter's activity. Thus, GATA3 was found to bind the site located at −954/−855 and to be a key regulator of abundant KLK1 expression in human keratinocyte.