Inactivation of the klotho gene in mice causes serious systemic disorders, resembling human aging. However, at the molecular level, its action mechanisms are not well understood. The stimulatory or inhibitory effects of cis- and trans-regulatory factors on the klotho gene expression are also still unclear. We studied the effects of intra- and extracellular factors on human klotho gene expression. For this purpose, pHKP-Luc and pHKP-GFP reporter vectors were constructed with the 2.1-kbp upstream region of human klotho, covering its promoter region, using luciferase and GFP genes as the reporter. A series of vectors that have deletions in the upstream region of the klotho gene were constructed to assay cis-acting factors. Deletion of some parts of the klotho gene upstream region significantly affected reporter gene expression in HEK293 cells. p16 and p53 proteins inhibited reporter luciferase expression under the control of human klotho promoter in a dose-dependent manner. Calcium and phosphate ions stimulated klotho expression. p21, PTH, IGF-1, and angiotensin-II had no significant effect on klotho expression in HEK293 cells.