Interferon epsilon (IFNε) is a type I IFN that is expressed constitutively in the female reproductive tract (FRT), and contributes to protection in models of sexually transmitted infections. Using multiple cell systems, including reporter cell lines and activated peripheral blood lymphocytes (PBLs), we show that recombinant IFNε impairs HIV infection at stage(s) post HIV entry and up to the translation of viral proteins. Consistent with this, IFNε upregulated a number of host cell restriction factors that block HIV at these stages of the replication cycle. The potency of IFNε induction of these HIV restriction factors was comparable to conventional type I IFNs, namely IFNα and IFNβ. IFNε also significantly reduced the infectivity of progeny virion particles likely by inducing expression of HIV restriction factors, such asIFITM3, which act at that stage of infection. Thus, our data demonstrate that human IFNε suppresses HIV replication at multiple stages of infection.