Of the 486,000 burn injuries that required medical treatment in the USA in 2016, 40,000 people were hospitalized, with > 3,000 fatalities. After burn injury, humans are at increased risk of sepsis and mortality from infections caused by Pseudomonas aeruginosa (PA), an opportunistic pathogen. We hypothesize that systemic events were initiated from the burn that increased the host’s susceptibility to PA. A non-lethal 10% total body surface area (TBSA), full-thickness flame burn was performed in CD-1 mice without and with subsequent PA (strain M2) infection. The LD50 for subcutaneous infection with PA M2 at the burn site immediately after the burn decreased by 6-logs with mortality occurring between 18 and 26 hours, compared with PA-infected mice without burn injury. Bacteria in distal organs were detected by 18 hours, concurrent with the onset of clinical symptoms. Serum pro-inflammatory cytokines (IL-6, IL-1?, IFN-?, and TNF-?) and the anti-inflammatory cytokine, IL-10, were first detected at 12 hours post-burn with infection and continued to increase until death. Directly after burn alone, serum levels of HMGB1, a danger-associated molecular pattern and TLR4 agonist, transiently increased to 50 ng/mL before returning to 20 ng/mL. Burn with PA infection increased serum HMGB1 concentrations >10-fold (250 ng/mL) at the time of death. This HMGB1-rich serum stimulated TLR4-mediated NF-?B activation in a TLR4-reporter cell line. Treatment of infected burned mice with P5779, a peptide inhibitor of HMGB1, increased the mean survival from 23 to 42 hours (P<0.0001). We conclude that the high level of serum HMGB1, which preceded the increase in pro-inflammatory cytokines, is associated with post-burn mortality.