With-no-lysine (K) kinase-4 (WNK4) is a serine/threonine protein kinase and plays a crucial role in the regulation of blood pressure and electrolyte homeostasis. Whether or not estrogen, an important regulator of physiologic functions, modulates human WNK4 (hWNK4) gene expression is unknown. In the current study, real-time PCR assay showed that 17β-estradiol (E(2)) with estrogen receptor alpha (ERα) suppressed the level of hWNK4 mRNA in cultured human embryo kidney 293 cells (HEK293). Luciferase activity analysis of the truncated hWNK4 promoters indicated that a regulatory region from -216 to -202 is essential for the basal transcriptional activity and response to E(2)/ERα. Using an electrophoresis mobility shift assay and a chromatin immunoprecipitation assay, we identified an activator protein-1 (AP-1) element at position -215/-205, to which AP-1 binding was enhanced by E(2)/ERα. Accordingly, AP-1 protein was increased by E(2)/ERα using Western blot analysis. Moreover, re-chromatin-immunoprecipitation and co-immunoprecipitation assays showed a direct interaction between ERα and AP-1 in HEK293 cells. In summary, these data document that E(2)/ERα regulates hWNK4 expression partly through AP-1 binding to the hWNK4 promoter.