Influenza virus is a worldwide global health concern causing seasonal morbidity, mortality, and economic burden. Chemotherapeutics is available however rapid emergence of drug resistant influenza strains has reduced their efficacy, thus there is a need to discover novel anti-viral agents. In this study, RNA interference (RNAi) was used to screen host genes required for influenza virus replication. One pro-influenza virus host gene identified was dual-specificity phosphatase cell division cycle 25 B (CDC25B). RNAi of CDC25B resulted in reduced influenza A virus replication, and a CDC25B small molecule inhibitor (NSC95397) inhibited influenza A virus replication in dose-dependent fashion. Viral RNA synthesis was reduced by NSC95397 in favor of increased interferon beta (IFNβ) expression, and NSC95397 was found to interfere with nuclear localization and chromatin association of NS1, an influenza virus protein. As NS1 has been shown to be chromatin-associated to suppress host transcription, it is likely that CDC25B supports NS1 nuclear function to hijack host transcription machinery in favor of viral RNA synthesis, a process that is blocked by NSC95397. Importantly, NSC95397 treatment protects mice against lethal influenza virus challenge. The findings establish CDC25B as a pro-influenza A host factor that may be targeted as a novel influenza A therapeutic strategy.