NKT cells are remarkably abundant in mouse liver. Compelling experimental evidence has suggested that NKT cells are involved in the pathogenesis of many liver diseases. Activation of NKT cells with α-galactosylceramide (α-GalCer) causes liver injury through mechanisms that are not well understood. We undertook studies to characterize the key pathways involved in α-GalCer–induced liver injury. We found that expression of the transcription factor IFN regulatory factor 1 (IRF-1) in mouse liver was dramatically upregulated by α-GalCer treatment. Neutralization of either TNF-α or IFN-γ inhibited α-GalCer–mediated IRF-1 upregulation. α-GalCer–induced liver injury was significantly suppressed in IRF-1 knockout mice or in wild-type C56BL/6 mice that received a microRNA specifically targeting IRF-1. In contrast, overexpression of IRF-1 greatly potentiated α-GalCer–induced liver injury. α-GalCer injection also induced a marked increase in hepatic inducible NO synthase expression in C56BL/6 mice, but not in IRF-1 knockout mice. Inducible NO synthase knockout mice exhibited significantly reduced liver injury following α-GalCer treatment. Finally, we demonstrated that both NKT cells and hepatocytes expressed IRF-1 in response to α-GalCer. However, it appeared that the hepatocyte-derived IRF-1 was mainly responsible for α-GalCer–induced liver injury, based on the observation that inhibition of IRF-1 by RNA interference did not affect α-GalCer–induced NKT cell activation. Our findings revealed a novel mechanism of NKT cell-mediated liver injury in mice, which has implications in the development of human liver diseases.