The renin-angiotensin system (RAS) is a major mediator of renal injury in diabetic nephropathy. Our previous studies have demonstrated that 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) plays a renoprotective role by suppressing the RAS, with renin and angiotensinogen (AGT) as the main targets. The mechanism whereby 1,25(OH)₂D₃) transcriptionally suppresses renin gene expression has been elucidated; however, how vitamin D regulates AGT remains unknown. Exposure of mesangial cells or podocytes to high glucose (HG, 30 mM) markedly stimulated AGT expression. In mesangial cells, the stimulation was inhibited by 1,25(OH)₂D₃) (20 nM) or NF-B inhibitor BAY 11-7082, suggesting the involvement of NF-B in HG-induced AGT expression and the interaction between 1,25(OH)₂D₃) and NF-B in the regulation. Plasmid pNF-B-Luc luciferase reporter assays showed that 1,25(OH)₂D₃) blocked HG-induced NF-B activity. EMSA and ChIP assays demonstrated increased p65/p50 binding to a NF-B binding site at -1734 in the AGT gene promoter, and the binding was disrupted by 1,25(OH)₂D₃) treatment. Overexpression of p65/p50 overcame 1,25(OH)₂D₃) suppression, and mutation of this NF-B binding site blunted 1,25(OH)₂D₃) suppression of the promoter activity. In mice lacking the vitamin D receptor, AGT mRNA expression in the kidney was markedly increased compared to wild-type mice, and AGT induction in diabetic mice was suppressed by treatment with a vitamin D analog. These data indicate that 1,25(OH)₂D₃) suppresses hyperglycemia-induced AGT expression by blocking NF-B-mediated pathway.