Background Our previous in vitro work showed that stored red blood cells (RBCs) increasingly suppress markers of innate immune function with increased storage time. This multicenter prospective observational study tests the hypothesis that a single RBC transfusion in critically ill children is associated with immune suppression as a function of storage time. Study Design and Methods Blood samples were taken immediately before and 24 (±6) hours after a single RBC transfusion ordered as part of routine care. Innate and adaptive immune function was assessed by ex vivo whole blood stimulation with lipopolysaccharide (LPS) and phytohemagglutinin, respectively. Monocyte HLA-DR expression, regulatory T cells, plasma interleukin (IL)-6, and IL-8 levels were also measured. Results Thirty-one transfused critically ill children and eight healthy controls were studied. Critically ill subjects had lower pretransfusion LPS-induced tumor necrosis factor-α production capacity compared to healthy controls, indicating innate immune suppression (p < 0.0002). Those who received RBCs stored for not more than 21 days demonstrated recovery of innate immune function (p = 0.02) and decreased plasma IL-6 levels (p = 0.002) over time compared to children transfused with older blood, who showed persistence of systemic inflammation and innate immune suppression. RBC storage time was not associated with changes in adaptive immune function. Conclusion In this pilot cohort of critically ill children, transfusion with older prestorage leukoreduced RBCs was associated with persistence of innate immune suppression and systemic inflammation. This was not seen with fresher RBCs. RBC transfusion had no short-term association with adaptive immune function. Further studies are warranted to confirm these findings in a larger cohort of patients.