Although the anti-tumor effect of IL-12 is mediated mostly by IFNgamma, which cell types most efficiently produce IFNgamma and therefore initiate or promote the anti-tumor effect of IL-12 has not been clearly determined. In the present study, we demonstrated hydrodynamic injection of the IL-12 gene led to prolonged IFNgamma production, NK-cell activation and complete inhibition of liver metastasis of CT-26 colon cancer cells in wild-type mice, but not in IFNgamma knockout mice. NK cells expressed higher levels of STAT4 and upon IL-12 administration displayed stronger STAT4 phosphorylation and IFNgamma production than non-NK cells. Adoptive transfer of wild-type NK cells into IFNgamma knockout mice restored IL-12-induced IFNgamma production, NK-cell activation and anti-tumor effect, whereas transfer of the same number of wild-type non-NK cells did not. In conclusion, NK cells are predominant producers of IFNgamma that is critical for IL-12 anti-tumor therapy.