Klotho is a senescence suppressor protein that, when overexpressed, extends the lifespan of mice. Klotho-disrupted mice exhibit atherosclerosis and endothelial dysfunction, which led us to investigate the effect of the Klotho protein on vascular inflammation, particularly adhesion molecule expression. In this study, human umbilical vein endothelial cells (HUVECs) were preincubated with Klotho protein and then exposed to tumor necrosis factor-α (TNF-α) or vehicle. Reverse transcription-PCR and Western blot analyses revealed that Klotho suppressed TNF-α-induced expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). NF-κB activation, IκB phosphorylation induced by TNF-α were also attenuated by Klotho protein administration. The inhibition of eNOS phosphorylation by TNF-α was reversed by Klotho. Furthermore, Klotho inhibited TNF-α-induced monocyte adhesion to HUVECs and suppressed adhesion molecule expression in an organ culture of the rat aorta. These results suggest that Klotho suppresses TNF-α-induced expression of adhesion molecules and NF-κB activation. Klotho may have a role in the modulation of endothelial inflammation.