In addition to its well-known role in mineral and skeletal homeostasis, 1,25-dihydroxyvitamin D3[1,25-(OH)2, D3] regulates the differentiation, growth and function of a broad range of immune system cells, including monocytes, dendritic cells, T and B lymphocytes. Vascular endothelial cells play a major role in the innate immune activation during infections, sepsis and transplant rejection; however, currently there are no data on the effect of 1,25-(OH)2 D3 on microbial antigen-induced endothelial cell activation. Here we show that 1,25-(OH)2 D3 pretreatment of human microvessel endothelial cells (HMEC) inhibited the enteric Gram-negative bacterial lipopolysaccharide (LPS) activation of transcription factor NF-κB and interleukin (IL)-6, IL-8 and regulated upon activation normal T cell exposed and secreted (RANTES) release. The effect of 1,25-(OH)2 D3 was not due to increased cell death or inhibition of endothelial cell proliferation. 1,25-(OH)2 D3 pretreatment of HMEC did not block MyD88-independent LPS-induced interferon (IFN)-β promoter activation. 1,25-(OH)2 D3 pretreatment of HMEC did not modulate Toll-like receptor 4 (TLR4) or MD-2 expression. These data suggest that 1,25-(OH)2 D3 may play a role in LPS-induced immune activation of endothelial cells during Gram-negative bacterial infections, and a suggest a potential role for 1,25-(OH)2 D3 and its analogues as an adjuvant in the treatment of Gram-negative sepsis.