Human cytomegalovirus virus (CMV) is a major cause of morbidity and mortality in immunocompromised individuals. Recently, a novel group of cytokines [interleukin (IL)-28A/B and IL-29, also termed interferon (IFN)-λs] has been described. Here, we demonstrate that intestinal epithelial cell (IEC) lines as well as murine and human colonic tissue express the IFN-λ receptor subunits IL-28R and IL-10R2. IL-28A and IL-29 binding to their receptor complex activates ERK-1/2 and stress-activated protein kinase/c-Jun NH2-terminal kinase MAPKs and Akt, resulting in increased IL-8 protein expression. IFN-λs also induce phosphorylation of signal transducer and activator of transcription 1 and significantly increase mRNA expression of suppressor of cytokine signaling 3 and the antiviral proteins myxovirus resistance A and 2′,5′-oligoadenylate synthetase. These signals result in an up to 83% reduction of cells positive for human CMV immediate-early protein after human CMV infection. In mice, IL-28A mRNA expression is upregulated after infection with murine CMV in vivo. Both IL-28A and IL-29 significantly decrease cell proliferation but have no effect on Fas-induced apoptosis. In conclusion, IECs express functional receptors for IFN-λs, which mediate antiviral and antiproliferative signals in IECs, suggesting a potential for therapeutic use in certain viral infections and as (antiproliferative) anticancer therapy.