We previously reported that TLR4(-/-) mice are refractory to mouse-adapted A/PR/8/34 (PR8) influenza-induced lethality and that therapeutic administration of the TLR4 antagonist Eritoran blocked PR8-induced lethality and acute lung injury (ALI) when given starting 2 days post infection. Herein we extend these findings: anti-TLR4- or -TLR2-specific IgG therapy also conferred significant protection of wild-type (WT) mice from lethal PR8 infection. If treatment is initiated 3 h before PR8 infection and continued daily for 4 days, Eritoran failed to protect WT and TLR4(-/-) mice, implying that Eritoran must block a virus-induced, non-TLR4 signal that is required for protection. Mechanistically, we determined that (i) Eritoran blocks high-mobility group B1 (HMGB1)-mediated, TLR4-dependent signaling in vitro and circulating HMGB1 in vivo, and an HMGB1 inhibitor protects against PR8; (ii) Eritoran inhibits pulmonary lung edema associated with ALI; (iii) interleukin (IL)-1β contributes significantly to PR8-induced lethality, as evidenced by partial protection by IL-1 receptor antagonist (IL-1Ra) therapy. Synergistic protection against PR8-induced lethality was achieved when Eritoran and the antiviral drug oseltamivir were administered starting 4 days post infection. Eritoran treatment does not prevent development of an adaptive immune response to subsequent PR8 challenge. Overall, our data support the potential of a host-targeted therapeutic approach to influenza infection.