Macrophages accumulate in kidney glomeruli and interstitium of patients with diabetic nephropathy in response to monocyte chemoattractant protein-1 (MCP-1); a chemokine produced by both tubular epithelial and mesangial cells (MCs). Vitamin D and its analogs have been shown to have renoprotective effects; however, there are few studies involving diabetic nephropathy. We explored mechanisms by which 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) can be renoprotective by measuring MCP-1 expression in MCs. Using a luciferase reporter assay, we found that high glucose (HG)-induced MCP-1 transcription and that this induction is blocked by 1,25(OH)₂D₃. Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that HG increased the p65/p50 binding to the two NF-κB sites within the promoter. This was suppressed by 1,25(OH)₂D₃, but this decrease was reversed by overexpression of p65. 1,25(OH)₂D₃ was found to stabilize IκBα leading to an inhibition of p65 translocation to the nucleus and subsequent reduction of NF-κB binding. In primary MCs prepared from vitamin D receptor knockout animals, basal MCP-1 levels were elevated but not affected by 1,25(OH)₂D_3. The analog paricalcitol inhibited the induction and activity of MCP-1 while ameliorating glomerulosclerosis in streptozotocin-diabetic mice. Our results suggest that 1,25(OH)₂D₃ might block hyperglycemia-induced renal injury by blunting NF-κB activation.