Influenza A virus (flu) is a respiratory tract pathogen causing high morbidity and mortality among the human population. NO is a cellular mediator involved in tissue damage through its apoptosis of target cells and resulting enhancement of local inflammation. Inducible NO synthase (iNOS) is involved in the production of NO following infection. Although NO is a key player in the development of exaggerated lung disease during flu infection, the underlying mechanism, including the role of NO in apoptosis during infection, has not been reported. Similarly, the mechanism of iNOS gene induction during flu infection is not well defined in terms of the host transactivator(s) required for iNOS gene expression. In the current study, we identified Kruppel-like factor 6 (KLF6) as a critical transcription factor essential for iNOS gene expression during flu infection. We also underscored the requirement for iNOS in inducing apoptosis during infection. KLF6 gene silencing in human lung epithelial cells resulted in the drastic loss of NO production, iNOS promoter-specific luciferase activity, and expression of iNOS mRNA following flu infection. Chromatin immunoprecipitation assay revealed a direct interaction of KLF6 with iNOS promoter during in vitro and in vivo flu infection of human lung cells and mouse respiratory tract, respectively. A significant reduction in flu-mediated apoptosis was noted in KLF6-silenced cells, cells treated with iNOS inhibitor, and primary murine macrophages derived from iNOS knockout mice. A similar reduction in apoptosis was noted in the lungs following intratracheal flu infection of iNOS knockout mice.