Klotho is an antiaging gene involved in the suppression of several age-related phenotypes, but few studies have examined the mechanism underlying the regulation of human Klotho gene expression. In this study, we investigated the transcriptional regulation of the Klotho gene by epidermal growth factor (EGF) in HEK293 human embryonic kidney cells. By using serial deletion constructs of the promoter, we identified a proximal 45 bp (-90 to -45) region responsible for EGF-induced promoter activity. The Egr-1-binding motif is located within this region. Forced expression of Egr-1 stimulated Klotho gene promoter activity. A point mutation in the Egr-1-binding motif abrogated promoter inducibility by EGF or ectopic Egr-1 expression. Knockdown of Egr-1 by expression of small interfering RNA (siRNA) attenuated EGF-induced Klotho promoter activity. Further analysis showed that the Ras/MEK/Erk signaling cascade is involved in EGF-induced activation of the Klotho promoter. We conclude that the Klotho gene is activated by EGF in HEK293 cells.