Natural killer T (NKT) cells play an important role in both innate and adaptive immunity. CD160, a glycosylphosphatidylinositol-anchored Immunoglobulin (Ig) domain protein that binds to HVEM (herpes virus entry mediator), is expressed on T cells and NKT cells. Although the inhibitory role of CD160 in antigen-specific CD8+ T cells is well documented in conditions of chronic antigen exposure, the role of CD160 in NKT cells have not been clearly understood. Therefore, we investigated the role of CD160 in NKT cells isolated from WT and CD160-deficient mice. Although CD160-/- mice showed no apparent developmental defects, they were more susceptible to Concanavalin A (Con A)-induced hepatitis than WT mice in vivo. While WT mice recovered from acute hepatitis, all CD160-/- mice died within 24 hours following Con A challenges. Serum AST and ALT levels were increased along with increased IL-4, IL-6, IFN-γ and TNF-α. Furthermore, NKT cells harvested from the liver of CD160-/- mice showed significantly higher levels of IFN-γ, TNF-α, and IL-4 upon in vitro challenges of α-Galactosylceramide (α-GalCer) or Concanavalin A (Con A) than those from WT mice. These results demonstrate that CD160 plays a critical role in the resolution of acute hepatitis and liver damage, and dysregulation of CD160 receptor signaling in NKT cells resulted in liver injury and death associated with uncontrolled cytokine responses in NKT cells. Therefore, we conclude that CD160 is a negative regulator of NKT cells.