RNA virus infection results in expression of type 1 IFNs, especially IFN-α/β, which play a crucial role in host antivirus responses. Type 1 IFNs are induced in a cell type-specific manner through TLR and RIG-I–like receptor pathways, both of which activate IFN regulatory factors (IRFs) and NF-κB transcription factors. Although NF-κB activation and association with the IFN-β promoter after RNA virus infection is well documented, our previous work showed that, surprisingly, NF-κB is not essential for IFN-β gene expression. Thus, the actual function of NF-κB in IFN-β expression and virus replication is not clear. In this study, we found Newcastle disease virus and vesicular stomatitis virus replication is enhanced in mouse embryonic fibroblasts (MEFs) lacking the NF-κB RelA subunit. Increased virus replication was traced to a specific requirement for RelA in early virus-induced IFN-β expression. At these time points, when IFN-β expression is~ 100-fold less than peak levels, impaired IFN-β production delayed IFN-induced gene expression, resulting in increased virus replication in RelA−/− MEFs. Importantly, our results show that RelA requirement is crucial only when IRF3 activation is low. Thus, high levels of activated IRF3 expression are sufficient for induction of IFN-β in RelA−/− MEFs, transcriptional synergism with the coactivator CREB-binding protein, and rescue of susceptibility to virus. Together, these findings indicate that NF-κB RelA is not crucial for regulating overall IFN-β production, as previously believed; instead, RelA is specifically required only during a key early phase after virus infection, which substantially impacts the host response to virus infection.