Aims: Influenza A virus (IAV), a major airborne pathogen, is closely associated with significant morbidity and mortality. The primary target for influenza virus replication is the respiratory epithelium, which reacts to infection by mounting a multifaceted antiviral response. A part of this mucosal host defense is the generation of reactive oxygen species (ROS) by NADPH oxidases. Duox1 and Duox2 are the main ROS-producing enzymes in the airway epithelium, but their contribution to mammalian host defense is still ill defined. Results: To gain a better understanding of Duox function in respiratory tract infections, human differentiated lung epithelial cells and an animal model were used to monitor the effect of epithelial ROS on IAV propagation. IAV infection led to coordinated up-regulation of Duox2 and Duox-mediated ROS generation. Interference with H₂O₂ production and ROS signaling by oxidase inhibition or H₂O₂ decomposition augmented IAV replication. A nuclear pool of Duox enzymes participated in the regulation of the spliceosome, which is critical for alternative splicing of viral transcripts and controls the assembly of viable virions. In vivo silencing of Duox increased the viral load on intranasal infection with 2009 pandemic H1N1 influenza virus. Innovation: This is the first study conclusively linking Duox NADPH oxidases with the antiviral mammalian immune response. Further, ROS generated by Duox enzymes localized adjacent to nuclear speckles altered the splicing of viral genes. Conclusion: Duox-derived ROS are host protective and essential for counteracting IAV replication. Antioxid. Redox Signal. 20, 2695–2709.