The herpes simplex virus 2 (HSV-2) virion host shutoff (vhs) protein is a ribonuclease contained in the virion tegument. vhs-deficient mutants of HSV-2 are profoundly attenuated in vivo, and we have previously shown that replication and virulence of vhs-deficient HSV-2 are largely restored to levels of wild-type virus in mice lacking the interferon alpha/beta receptor (IFNalphabetaR(-/-)). This result demonstrated that HSV-2 vhs interferes with the type I IFN response, but whether vhs inhibits production of type I IFN or synthesis or function of key mediators of the IFN-induced antiviral state was not clear. Here we address these questions using primary murine embryonic fibroblasts (MEFs), which produce and respond to IFNalphabeta. The vhs-deficient HSV-2 strain 333d41 replicated similarly to wild-type virus (333 clone SB5) and vhs rescue virus (333d41(R)) after infection of MEFs at high moi, but at low moi, 333d41 replication was severely attenuated, recapitulating the attenuated phenotype of vhs-deficient HSV-2 in vivo. Replication of 333d41 at low moi was restored to levels of wild-type virus in MEFs lacking the IFNalphabeta receptor or when IFNalphabeta was neutralized, thus establishing the IFNalphabeta response as the sole mechanism attenuating vhs-deficient HSV-2 replication in MEFs. MEFs infected with 333d41 produced >50-fold more IFNalphabeta than cells infected with 333 and 333d41(R). Pretreatment of MEFs with type I IFN inhibited replication of 333d41 more than 333 and 333d41(R), indicating that vhs also interferes with activation of the IFNalphabeta-induced antiviral response. We therefore examined vhs interference with PKR and RNase L, two key mediators of the IFNalphabeta response. 333d41 replication was restored to wild-type levels after low moi infection of PKR(-/-) and RNase L(-/-) MEFs, and was not inhibited in PKR(-/-) MEFs pretreated with IFNalpha. Together, these observations indicate that HSV-2 vhs is a broad and potent countermeasure to the IFN-mediated antiviral response in IFN-naïve and -sensitized MEFs.