Technical Abstract: While pandemic 2009 H1N1 influenza A viruses were responsible for numerous severe infections in humans, these viruses do not typically cause corresponding severe disease in mammalian models. However, the generation of a virulent 2009 H1N1 virus following serial lung passage in mice has allowed for the modeling of human lung pathology in this species. Genetic determinants of mouse-adapted 2009 H1N1 viral pathogenicity have been identified, but the molecular and signaling characteristics of the host response following infection with this adapted virus have not been described. Here, we compared the gene-expression response following infection of mice with A/CA/04/2009 (CA/04) or the virulent mouse-adapted strain (MA-CA/04). Microarray analysis revealed that increased pathogenicity of MA CA/04 was associated with: 1) early and sustained inflammatory and interferon response that could be driven in part by interferon regulatory factors (IRFs) and increased nuclear factor kappa B (NFKB) activation, as well as inhibition of the negative regulator tripartite motif-containing 24 (TRIM24); 2) early and persistent infiltration of immune cells, including inflammatory macrophages; and 3) the absence of activation of lipid metabolism later in infection, that may be mediated by nuclear receptors inhibition, with pro inflammatory consequences. Further investigation of these signatures in the host response to other H1N1 viruses of varied pathogenicity confirmed their general relevance for virulence of influenza virus and suggested that lung response to MA-CA/04 virus was similar to that following lethal H1N1 r1918 influenza virus. This study links for the first time differential activation of IRFs, nuclear receptors, and macrophage infiltration with influenza virulence in vivo.