Herein, we report that Autographa californica nucleopolyhedrovirus, a member of the Baculoviridae family, is capable of stimulating antiviral activity in mammalian cells. Baculoviruses are not pathogenic to mammalian cells. Nevertheless, live baculovirus is shown here to induce interferons (IFN) from murine and human cell lines and induces in vivo protection of mice from encephalomyocarditis virus infection. Monoclonal antibodies specific for the baculovirus envelope gp67 neutralize baculovirus-dependent IFN production. Moreover, UV treatment of baculovirus eliminates both infectivity and IFN-inducing activity. In contrast, the IFN-inducing activity of the baculovirus was unaffected by DNase or RNase treatment. These data demonstrate that IFN production can be induced in mammalian cells by baculovirus even though the cells fail to serve as a natural host for an active viral infection. Baculoviruses, therefore, provide a novel model in which to study at least one alternative mechanism for IFN induction in mammalian cells.