IL-28B is a member of the newly discovered type III IFN family and exhibits unique antiviral properties compared with other family members. NK cells play a critical role in defending against viruses; however, little is known about the role of IL-28B in NK cell function. In a mouse model of influenza A virus (mouse adapted influenza A/PR/8/34 strain) infection, long-term overexpression of IL-28B induced by hepatocyte-specific gene delivery exerted a strong antiviral effect in the presence of NK cells. In IL-28B-overexpressing wild-type mice, the percentages and absolute numbers of NK cells in the spleen, liver, and lung were markedly increased, with higher proliferation and accelerated NK cell maturation based on phenotypes staining with CD11b and CD27 or CD11b and KLRG1. Furthermore, the effect of IL-28B on NK cells was macrophage dependent, as confirmed in an in vitro coculture assay and in in vivo macrophage- or alveolar macrophage-depletion experiments. Transwell studies demonstrated that CFSE-labeled NK cell proliferation was driven, in a dose-dependent manner, by unknown soluble factor(s) secreted by IL-28B-stimulated alveolar macrophages, without requiring direct cell-cell contact. An understanding of the NK cell-promoting features of IL-28B will facilitate future clinical application of this cytokine.