Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common known human genetic polymorphism. This study tested the hypothesis whether G6PD deficiency worsens sepsis-induced erythrocyte dysfunction. Sepsis (24h) was induced by cecal ligation and puncture in wild type (WT) and G6PD deficient (G6PD activity 15% of WT) mice. Erythrocyte responses were tested in whole blood as well as in subpopulations of circulating erythrocytes. Whereas erythrocyte deformability was similar in unchallenged deficient and wt animals, sepsis decreased erythrocyte deformability that was more pronounced in deficient than WT animals. Sepsis also resulted in anaemia and hemolysis in deficient animals compared to WT. Mean corpuscular hemoglobin content and erythrocyte deformability decreased in younger erythrocyte subpopulations from septic deficient animals compared to WT. Sepsis decreased the reduced/oxidized glutathione-ratio in erythrocytes from both deficient and WT animals however, plasma glutathione increased more in deficient than WT animals. Erythrocyte content of band 3 associated with the cytoskeleton was elevated in deficient erythrocyte compared to WT. The antioxidant, N-acetyl-cysteine in vivo alleviated the sepsis induced-decrease in erythrocyte deformability in deficient animals compared to sham-operated controls. This study demonstrates that a mild degree of G6PD deficiency (comparable to the human Class III G6PD deficiencies) worsens erythrocyte dysfunction during sepsis. Increased erythrocyte rigidity and tendency for hemolysis together with alterations in band 3-spectrin interactions may contribute to the immuno-modulatory effects of G6PD deficiency observed after major trauma and infections in humans.