Obesity is associated with hyperleptinemia and endothelial dysfunction. Hyperleptinemia has been reported to induce both oxidative stress and inflammation by increasing reactive oxygen species production. The objective of this study was to determine the effects of 1,25-dihydroxycholecalciferol [1,25(OH)D] against leptin-induced oxidative stress and inflammation in human endothelial cells. Small interfering RNA (siRNA) were used to knock down the expression of vitamin D receptor (VDR) in human umbilical vein endothelial cells (HUVECs). HUVECs were pretreated for 4 h with physiologic (10 M) or supraphysiologic (10 M) concentrations of 1,25(OH)D and exposed to leptin (10 ng/mL). Superoxide anion production and translocation of nuclear factor (erythroid-derived 2)-like 2 (NRF2) and nuclear transcription factor κB (NF-κB) subunit p65 to the nucleus and the activation of their target genes were quantified. Pretreatment of HUVECs with 1,25(OH)D prevented the leptin-induced increase in superoxide anion production ( < 0.05). Pretreatment with 1,25(OH)D further increased NRF2 translocation to the nucleus (by 3-fold; < 0.05) and increased mRNA expression of superoxide dismutase 2 (; by 2-fold), glutathione peroxidase (; by 3-fold), NAD(P)H dehydrogenase (quinone) 1 (; by 4-fold), and heme oxygenase 1 (; by 2-fold) ( < 0.05). Leptin doubled the translocation of NF-κB ( < 0.05) to the nucleus and increased ( < 0.05) the upregulation of vascular inflammatory mediators such as monocyte chemoattractant protein 1 (; by 1-fold), transforming growth factor β ( β by 1-fold), and vascular cell adhesion molecule 1 (; by 4-fold) ( < 0.05), which were prevented ( < 0.05) by pretreatment with 1,25(OH)D Protective effects of 1,25(OH)D were confirmed to be VDR dependent by using VDR siRNA. Pretreatment with 1,25(OH)D in the presence of a high concentration of leptin has a beneficial effect on HUVECs through the regulation of mediators of antioxidant activity and inflammation.