Type I interferon (IFN), which includes the IFN-α and -β subtypes, plays an essential role in host defense against influenza A virus. However, the relative contribution of IFN-β remains unresolved. In mice, type I IFN is effective against influenza viruses only if the IFN-induced resistance factor Mx1 is present, though most inbred mouse strains, including the recently developed IFN-β-deficient mice, bear only defective Mx1 alleles. We therefore generated IFN-β-deficient mice carrying functional Mx1 alleles (designated Mx-BKO) and compared them to either wild-type mice bearing functional copies of both IFN-β and Mx1 (designated Mx-wt) or mice carrying functional Mx1 alleles but lacking functional type I IFN receptors (designated Mx-IFNAR). Influenza A virus strain SC35M (H7N7) grew to high titers and readily formed plaques in monolayers of Mx-BKO and Mx-IFNAR embryo fibroblasts which showed no spontaneous expression of Mx1. In contrast, Mx-wt embryo fibroblasts were found to constitutively express Mx1, most likely explaining why SC35M did not grow to high titers and formed no visible plaques in such cells. In vivo challenge experiments in which SC35M was applied via the intranasal route showed that the 50% lethal dose was about 20-fold lower in Mx-BKO mice than in Mx-wt mice and that virus titers in the lungs were increased in Mx-BKO mice. The resistance of Mx-BKO mice to influenza A virus strain PR/8/34 (H1N1) was also substantially reduced, demonstrating that IFN-β plays an important role in the defense against influenza A virus that cannot be compensated for by IFN-α.