The NKT cell ligand alpha-galactosylceramide and its synthetic homologue KRN7000 stimulate rapid and copious secretion of IFN-gamma and TNF-alpha release, both of which are key mediators of LPS-induced shock. We showed that KRN7000, injected before or within 2 h after LPS challenge, was able to prevent endotoxic shock. KRN7000 induced survival when the mice were injected 6, 9, or 12 days before the first injection of LPS, and this protective effect was associated with reduction upon subsequent challenge in the levels of IFN-gamma, TNF-alpha, MCP-1, and an increase of IL-10. Further analysis showed that the animals treated with KRN7000 prior to LPS challenge had lower numbers of F4/80+, NKT, and NK cells and lower percentages of NKT cells that stained for intracytoplasmic IFN-gamma when compared with mice that were not treated with KRN7000. When MCP-1 was injected in KRN7000-treated mice, the lethal effect of LPS challenge was restored, and the numbers of F4/80+, NKT, and NK cells increased to levels similar to those in untreated mice following LPS challenge. Taken together, our data demonstrated that KRN7000, injected from 6 to 12 days before the first administration of LPS, prevented endotoxin shock by inhibiting IFN-gamma, TNF-alpha, and MCP-1 release.