1,25-(OH)2 vitamin D3 is important for calcium homeostasis and cell differentiation. The key enzyme for the activation of liver-derived 25(OH) vitamin D3 is 25-hydroxyvitamin D3 1α-hydroxylase. It is expressed mainly in the kidney but also in peripheral tissues. A 1413-bp fragment of the 1α-hydroxylase promoter was cloned into luciferase vectors pGL2basic and pGL3basic. Sequence analyses revealed four base exchanges and three base deletions compared with the published sequence which were identically found in five control persons. In silico promoter analyses revealed 17 putative nuclear factor (NF)κB sites, 10 of which were found to bind NFκB in EMSA experiments. Cotransfection of NFκB p50 and p65 subunits resulted in dramatic reduction of the promoter activity of the full-length construct as well as a series of 5′-deletion constructs. Deletion of the farmost 3′-situated NFκB-responsive element almost abolished NFκB responsiveness. Treatment of human embryonic kidney 293 cells with sulfasalazine, a NFκB inhibitor, resulted in enhanced 1α-hydroxylase mRNA production. Down-regulation of 1α-hydroxylase promoter through NFκB signaling may contribute to the pathogenesis of inflammation-associated osteopenia/osteoporosis.