Both free alpha-galactosylceramide (αGalCer) and αGalCer-loaded dendritic cells (DCG) activate invariant natural killer T (iNKT) cells to varying degrees, with αGalCer inducing liver injury. We sought to evaluate liver injury by these two pathways.Mice were injected with αGalCer or DCG followed by analysis of serum alanine transaminase (ALT) activity levels, mortality and liver function.While ALT levels were elevated after DCG in a tumor necrosis factor (TNF)-α-dependent manner, DCG did not cause lethal injury. More serious injury of liver CD31-positive endothelial cells (CD31(+) EC) was observed in mice treated with αGalCer than with DCG. Furthermore, liver CD31(+) EC of αGalCer-treated mice induced naïve liver lymphocytes to produce TNF-α.DCG treatment did not induce lethal liver injury. CD31(+) EC may play an antigen-presenting role to iNKT cells after αGalCer treatment and may be a cause of lethal injury.