Obesity is a global pandemic characterized by multiple comorbidities, including cardiovascular and metabolic diseases. The aim of this study was to define the associations between blood donor body mass index (BMI) and RBC measurements of metabolic stress and hemolysis. The associations between donor BMI (<25 kg/m2 , normal weight; 25-29.9 kg/m2 , overweight; and ≥30 kg/m2 , obese) and hemolysis (storage, osmotic, and oxidative; n = 18 donors) or posttransfusion recovery (n = 14 donors) in immunodeficient mice were determined in stored leukocyte-reduced RBC units. Further evaluations were conducted using the National Heart, Lung, and Blood Institute RBC-Omics blood donor databases of hemolysis (n = 13 317) and metabolomics (n = 203). Evaluations in 18 donors revealed that BMI was significantly (P < 0.05) and positively associated with storage and osmotic hemolysis. A BMI of 30 kg/m2 or greater was also associated with lower posttransfusion recovery in mice 10 minutes after transfusion (P = 0.026). Multivariable linear regression analyses in RBC-Omics revealed that BMI was a significant modifier for all hemolysis measurements, explaining 4.5%, 4.2%, and 0.2% of the variance in osmotic, oxidative, and storage hemolysis, respectively. In this cohort, obesity was positively associated (P < 0.001) with plasma ferritin (inflammation marker). Metabolomic analyses on RBCs from obese donors (44.1 ± 5.1 kg/m2 ) had altered membrane lipid composition, dysregulation of antioxidant pathways (eg, increased oxidized lipids, methionine sulfoxide, and xanthine), and dysregulation of nitric oxide metabolism, as compared to RBCs from nonobese (20.5 ± 1.0 kg/m2 ) donors. Obesity is associated with significant changes in RBC metabolism and increased susceptibility to hemolysis under routine storage of RBC units. The impact on transfusion efficacy warrants further evaluation.