In experimental pneumonia we found that transfused older blood increased mortality and lung injury that was associated with increased in vivo hemolysis and elevated plasma cell-free hemoglobin (CFH), non-transferrin-bound iron (NTBI), and plasma labile iron (PLI) levels. In this study, we additionally analyze identically treated animals that received lower or higher bacterial doses. Study Design and Methods Two-year-old purpose-bred beagles (n=48) challenged intrabronchially with Staphylococcus aureus (0 [n=8], 1.0×109 [n=8], 1.25×109 [n=24], and ≥1.5×109 [n=8]colony-forming units/kg) were exchange transfused with either 7- or 42-day-old canine universal donor blood (80mL/kg in four divided doses). Results The greater increases in CFH with older blood over days after exchange proved relatively independent of bacterial dose. The lesser increases in CFH observed with fresher blood were bacterial dose dependent potentially related to bacterial hemolysins. Without bacterial challenge, levels of CFH, NTBI, and PLI were significantly higher with older versus fresher blood transfusion but there was no significant measurable injury. With higher-dose bacterial challenge, the elevated NTBI and PLI levels declined more rapidly and to a greater extent after transfusion with older versus fresher blood, and older blood was associated with significantly worse shock, lung injury, and mortality. Conclusion The augmented in vivo hemolysis of transfused older red blood cells (RBCs) appears to result in excess plasma CFH and iron release, which requires the presence of established infection to worsen outcome. These data suggest that transfused older RBCs increase the risks from infection in septic subjects.