Type 1 IFNs (IFN-αβ) play pivotal roles in the host antiviral response and in TLR-induced signaling. IFN regulatory factor (IRF) and NF-κB transcription factors are thought to be crucial for virus-induced mRNA expression of IFN-β. Although recent studies have demonstrated essential roles for IRF3 and IRF7, the definitive role of NF-κB factors in IFN-β (or IFN-α) expression remains unknown. Using mice deficient in distinct members of the NF-κB family, we investigated NF-κB function in regulating type 1 IFN expression in response to Sendai virus and Newcastle disease virus infection. Surprisingly, IFN-β and IFN-α expression was strongly induced following virus infection of mouse embryonic fibroblasts (MEFs) from p50−/−, RelA/p65−/−, cRel−/−, p50−/− cRel−/−, and p50−/− RelA−/− mice. Compared with wild-type MEFs, only RelA−/− and p50−/− RelA−/− MEFs showed a modest reduction in IFN-β expression. To overcome functional redundancy between different NF-κB subunits, we expressed a dominant-negative IκBα protein in p50−/− RelA−/− MEFs to inhibit activation of remaining NF-κB subunits. Although viral infection of these cells failed to induce detectable NF-κB activity, both Sendai virus and Newcastle disease virus infection led to robust IFN-β expression. Virus infection of dendritic cells or TLR9-ligand CpG-D19 treatment of plasmacytoid dendritic cells from RelA−/− or p50−/− cRel−/− mice also induced robust type 1 IFN expression. Our findings therefore indicate that NF-κB subunits p50, RelA, and cRel play a relatively minor role in virus-induced type 1 IFN expression.