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, constitutively expressed IFNϵ has suggested a function for IFNs in reproductive tract homeostasis and protection from infections, but its intrinsic activities are untested. We report here the expression, purification, and functional characterization of murine IFNϵ (mIFNϵ). Recombinant mIFNϵ (rmIFNϵ) exhibited an α-helical fold characteristic of type I IFNs and bound to IFNα/β receptor 1 (IFNAR1) and IFNAR2, but, unusually, it had a preference for IFNAR1. Nevertheless, rmIFNϵ induced typical type I IFN signaling activity, including STAT1 phosphorylation and activation of canonical type I IFN signaling reporters, demonstrating that it uses the JAK-STAT signaling pathway. We also found that rmIFNϵ induces the activation of T, B, and NK cells and exhibits antiviral, antiproliferative, and antibacterial activities typical of type I IFNs, albeit with 100-1000-fold reduced potency compared with rmIFNα1 and rmIFNβ. Surprisingly, although the type I IFNs generally do not display cross-species activities, rmIFNϵ exhibited high antiviral activity on human cells, suppressing HIV replication and inducing the expression of known HIV restriction factors in human lymphocytes. Our findings define the intrinsic properties of murine IFNϵ, indicating that it distinctly interacts with IFNAR and elicits pathogen-suppressing activity with a potency enabling host defense but with limited toxicity, appropriate for a protein expressed constitutively in a sensitive mucosal site, such as the reproductive tract.