Transfused blood may have immunomodulatory and proinflammatory effects. We report the first randomized study exploring whether leukoreduced red blood cell (RBC) transfusion increases circulating proinflammatory mediators, markers of neutrophil activation, and the acute-phase response in critically ill adults. Eighty-four patients were recruited from six general intensive care units in the United Kingdom as part of a laboratory study nested within a parallel-group randomized trial comparing restrictive and liberal leukoreduced RBC transfusion strategies in critically ill patients aged more than 55 years with measured hemoglobin concentrations of not more than 90 g/L (ClinicalTrials.gov NCT00944112). Forty-one patients received transfusion and 43 did not receive transfusion. Plasma was sampled at baseline, 6 hours, and 24 hours after randomization or transfusion, and concentrations of interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12-p70, interferon-γ, tumor necrosis factor-α, human neutrophil elastase, soluble L-selectin, and C-reactive protein were measured using cytokine bead array analysis and an enzyme-linked immunosorbent assay. Patients who received transfusion did not have significantly different inflammatory biomarker plasma concentrations at the time points compared to those who did not receive transfusion, with the exception of IL-8 concentrations at 24 hours, which were reduced in the transfused group (p = 0.02). After adjustment for baseline inflammatory biomarker concentrations, there were no significant differences between patients who received transfusion and those who did not. Concentrations of measured biomarkers were not significantly increased during the first 24 hours after leukoreduced RBC transfusion. These data do not support the contention that leukoreduced RBC transfusion is associated with a proinflammatory response in the general adult critically ill population.