The type I interferons (IFNs) are a family of cytokines with diverse biological activities including antiviral, antiproliferative, and immunoregulatory functions. The discovery of the hormonally-regulated IFNε, which is constitutively expressed in the female reproductive tract, suggested a role for IFNs in both homeostasis and protection from infection at this unique site; however the intrinsic properties of IFNε were yet to be determined. We generated a recombinant form of murine (m)IFNε and report here its functional characterization. Recombinant mIFNε exhibited an α-helical fold, characteristic of other type I IFNs, and showed affinity for the extracellular domains (ECD) of murine IFN alpha/beta receptor (IFNAR)1-ECD and IFNAR2-ECD, albeit with an uncharacteristic preference for IFNAR1-ECD. Nevertheless, mIFNε induced typical type I IFN signaling activity, including STAT1 phosphorylation and activation of canonical type I IFN signaling reporters, demonstrating that it utilizes the canonical JAK/STAT signaling pathway. We also found that, similar to other type I IFNs, mIFNε exhibited antiviral, antiproliferative, and antibacterial activities, albeit with 100- to 1000-fold reduced potency compared with mIFNα1 and mIFNβ. mIFNε was also demonstrated to upregulate a lymphocyte activation marker (CD69) on T, B, and NK cells, again at reduced efficacy compared to mIFNα1 and mIFNβ. Surprisingly, although type I IFNs generally do not display cross-species activities, mIFNε exhibited an affinity for human IFNAR1-ECD and IFNAR2-ECD, and high antiviral activity on human cells, suppressing HIV replication and inducing the expression of known HIV-restriction factors in primary human lymphocytes. Our findings define the intrinsic properties of mIFNε, indicating that it distinctly interacts with IFNAR and elicits pathogen-suppressing activity with a potency enabling host defence, but with limited toxicity, appropriate for a protein expressed constitutively in a sensitive mucosal site, such as the reproductive tract.