Acetaminophen (APAP) overdose causes severe, fulminant liver injury. The underlying mechanism of APAP‐induced liver injury (AILI), studied by a murine model, displays similar characteristics of injury as those observed in patients. Previous studies suggest that aside from APAP‐induced direct damage to hepatocytes, the hepatic innate immune system is activated and may contribute to the overall pathogenesis of AILI. The current study employed the use of two murine natural killer (NK) cells with T‐cell receptor (NKT) cell knockout models (CD1d^−/− and Jα18^−/−) to elucidate the specific role of NKT cells in AILI. Compared to wild‐type (WT) mice, NKT cell‐deficient mice were more susceptible to AILI, as indicated by higher serum alanine transaminase levels and mortality. Increased levels of cytochrome P450 2E1 (CYP2E1) protein expression and activities, which resulted in increased APAP protein adduct formation, were observed in livers of APAP‐treated NKT cell‐deficient mice, compared to WT mice. Compared to WT mice, starvation of NKT cell‐deficient mice induced a higher increase of ketone bodies, which up‐regulate CYP2E1 through protein stabilization. Conclusion: Our data revealed a novel role of NKT cells in regulating responses to starvation‐induced metabolic stress. Elevated ketone body production in NKT cell‐deficient mice resulted in increased CYP2E1‐mediated APAP biotransformation and susceptibility to AILI. (HEPATOLOGY 2013)