Although we have previously demonstrated that IL-12 stimulation increases the number of hepatic natural killer (NK) T (NKT) cells and enhances liver injury during the early phase of liver regeneration, the role of NKT cells has remained unknown. We therefore evaluated the influence of NKT cells activated by IL-12 or by α-galactosylceramide (α-GalCer) on murine liver regeneration using Vα 14 NKT knockout (Jα 281−/−) mice. Levels of serum alanine aminotransferase (sALT) 24 hours after partial hepatectomy were enhanced in Jα 281+/+ but not in Jα 281−/− mice by both procedures. Hepatic NKT cells expressed considerably more interferon (IFN) γ and tumor necrosis factor α (TNF-α) messenger RNA (mRNA) after stimulation with both factors in Jα 281+/+ mice. Either anti-IFN-γ or TNF-α antibody inhibited the enhancement of liver injury. Furthermore, recombinant TNF-α injection similarly caused injury in hepatectomized livers of both Jα 281+/+ and Jα 281−/− mice; indeed, adoptively transferred TNF-α+/+ NKT cells enhanced liver injury after hepatectomy in TNF-α knockout mice. TNF receptor expressions on hepatocytes increased and peaked 24 hours after partial hepatectomy. In conclusion, simultaneous TNF-α synthesis and high levels of TNF receptor expression on hepatocytes cause severe liver damage by activated NKT cells during liver regeneration.