IFN-lambda 1, -lambda 2 and -lambda 3 have been discovered as the latest members of the class II cytokine family and shown to possess antiviral activity. Murine B16 melanoma and Colon26 cancer cells were transduced with mouse IFN-lambda to determine whether IFN-lambda possesses antitumor activity. Overexpression of IFN-lambda induced cell surface MHC class I expression and Fas/CD95 Ag, induced significant caspase-3/7 activity, and increased p21(Waf1/Cip1) and dephosphorylated Rb (Ser(780)) in B16 cells in vitro. IFN-lambda expression in tumor cell lines markedly inhibited s.c. and metastatic tumor formation in vivo compared with mock transfections (p < 0.05). Moreover, IFN-lambda expression induced lymphocytic infiltrates, and an Ab-mediated immune cell depletion assay showed that NK cells were critical to IFN-lambda-mediated tumor growth inhibition. Hydrodynamic injection of IFN-lambda cDNA successfully targeted liver metastatic foci of Colon26 cells, and moderately decreased the mortality of mice with tumors. IFN-lambda overexpression in the liver increased NK/NKT cells and enhanced their tumor-killing activity, and suggested the activation of innate immune responses. Thus, IFN-lambda induced both tumor apoptosis and NK cell-mediated immunological tumor destruction through innate immune responses. These findings suggested that local delivery of IFN-lambda might prove a useful adjunctive strategy in the clinical treatment of human malignancies.