1,8-Cineole (also known as eucalyptol) is a monoterpene that occurs naturally in many aromatic plants, 1,8-cineole has been reported to ameliorate dysfunction of endothelial cells. However, the mechanism of action of 1,8-cineole is incompletely understood. We investigated the protective effect of 1,8-cineole on lipopolysaccharide (LPS)-induced human umbilical vein endothelial cell (HUVEC) injury and the underlying mechanisms. HUVECs were preincubated with 1,8-cineole for 1.5 h, then exposed to LPS for 12 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase leakage assays showed 1,8-cineole reduced LPS-induced HUVEC injury significantly. Results from enzyme linked immunosorbent assays revealed that 1,8-cineole suppressed LPS-induced secretion of interleukin-6 and interleukin-8, and recovered nitric oxide to normal levels. 1,8-Cineole decreased phosphorylation of nuclear factor-kappa B (NF-κB) p65 and expression of inducible nitric oxide synthase, and simultaneously improved protein levels of endothelial nitric oxide synthase. Immunofluorescence confirmed 1,8-cineole moderates nuclear translocation of NF-κB. These results suggest that 1,8-cineole ameliorates HUVEC dysfunction significantly, and that this effect at least involves NF-κB suppression.