Two-year-old purpose-bred beagles (n = 24) infected with Staphylococcus aureus pneumonia were randomized in a blinded fashion for exchange transfusion with either 7- or 42-day-old canine universal donor blood (80 mL/kg in 4 divided doses). Older blood increased mortality (P = .0005), the arterial alveolar oxygen gradient (24-48 hours after infection; P ≤ .01), systemic and pulmonary pressures during transfusion (4-16 hours) and pulmonary pressures for ∼ 10 hours afterward (all P ≤ .02). Further, older blood caused more severe lung damage, evidenced by increased necrosis, hemorrhage, and thrombosis (P = .03) noted at the infection site postmortem. Plasma cell–free hemoglobin and nitric oxide (NO) consumption capability were elevated and haptoglobin levels were decreased with older blood during and for 32 hours after transfusion (all P ≤ .03). The low haptoglobin (r = 0.61; P = .003) and high NO consumption levels at 24 hours (r = −0.76; P < .0001) were associated with poor survival. Plasma nontransferrin-bound and labile iron were significantly elevated only during transfusion (both P = .03) and not associated with survival (P = NS). These data from canines indicate that older blood after transfusion has a propensity to hemolyze in vivo, releases vasoconstrictive cell-free hemoglobin over days, worsens pulmonary hypertension, gas exchange, and ischemic vascular damage in the infected lung, and thereby increases the risk of death from transfusion. •In canine S aureus pneumonia, first randomized blinded trial showing blood transfused at end of storage period increases mortality.•Increased in vivo hemolysis, cell-free hemoglobin, pulmonary hypertension, tissue damage, and gas exchange abnormalities each contributed.