Inflammation and endothelial cells contribute to the pathogenesis of various cardiovascular diseases (CVDs). Vitamin D is associated with a decreased risk of inflammation related diseases including CVDs. However, the links between vitamin D, endothelial cells and vascular inflammation remain to be elucidated. In this study, we investigated the anti-inflammatory effect of vitamin D in lipopolysaccharide (LPS)-activated endothelial cells and the related signaling pathways. 1,25(OH)₂D₃ treatment significantly suppressed LPS-induced upregulation of COX-2, TNF-α, IL-6 and PGE2 in Human umbilical vein endothelial cells (HUVECs). 1,25(OH)₂D₃ inhibited LPS-stimulated phosphorylation of Akt at S473 and T308, IκBα phosphorylation, and nuclear accumulation of NF-κB. Blocking PI3K signaling abolished the inhibitory effect of 1,25(OH)₂D₃ on LPS-induced increase of Akt S473 phosphorylation and COX-2 expression. Blocking NF-κB signaling blunted the suppression effect of 1,25(OH)₂D₃ on IκBα phosphorylation, NF-κB accumulation in the nucleus and COX-2 expression. These results indicate that 1,25(OH)₂D₃ suppresses inflammatory response in LPS-induced HUVECs through PI3K/Akt/NF-κB signaling pathway.