Herein we report the generation of Mouse monoclonal antibodies (mAbs) specific for the IFNAR-1 subunit of the mouse interferon-α/β (IFN-α/β) receptor (MAR1 mAbs) that block type I IFN receptor signaling and biologic response induction in vitro and in vivo. These mAbs were generated from Ifnar1-/- mice immunized by in vivo hydrodynamic transfection with a plasmid encoding the extracellular domain (ECD) of murine IFNAR-1. All MAR1 mAbs bound native receptor expressed on cell surfaces and immunoprecipitated IFNAR-1 from solubilized cells, and two mAbs also detected IFNAR-1 by Western blot analysis. In vitro, the mAbs prevented ligand-induced intracellular signaling and induction of a variety of type I IFN-induced biologic responses but had no effect on IFN-γ-induced responses. The most effective in vitro blocker, MAR1-5A3, also blocked type I IFN-induced antiviral, antimicrobial, and antitumor responses in vivo. We also explored whether murine IFNAR-1 surface expression required the presence of Tyk2. In contrast to Tky2-deficient human cell lines, comparable IFNAR-1 expression was found on primary cells derived either from wild-type or Tyk2-/- mice. These mAbs represent much needed tools to more clearly elucidate the biochemistry, cell biology, and physiologic function of the type I IFNs and their receptor in mediating host-protective immunity and immunopathology.