Human red blood cells (RBCs) have high abundance in blood tissue, usually 40-50% v/v. For the in vivo administered biomedical materials in contact with blood tissue, the RBCs are the major (in most cases, undesired) targets encountered. The interaction of the biomaterials with the RBCs will unavoidably occur, affecting the structure and function of the RBCs and then the whole organism. For the clinical applications of biomaterials, this effect should be clearly elucidated since it may cause acute or chronic harm to the organism. Moreover, the RBC-based experimental results could be extended to other tissue cells to a great extent. In this study, the effect of the branched polyethyleneimines (BPEIs) as a gene carrier on the structure and function of human RBCs was studied by using different molecular weights of the BPEIs. Specifically, the RBC aggregation and lysis induced by the BPEIs were first studied; then, the structural and conformational change of hemoglobin in the presence of the BPEIs was examined by using UV-vis, fluorescence, and circular dichroism spectroscopy. Furthermore, the oxygen-carrying function of the RBCs in the presence of the BPEIs was evaluated by measuring the 2,3-diphosphoglycerate (2,3-DPG) level and oxygen-dissociation curves. The results showed that the BPEIs with certain molecular weights at certain concentrations could cause RBC aggregation and lysis, alter the structure and conformation of hemoglobin, and impair the oxygen-delivery function of the RBCs. These data provide valuable information for the molecular design and clinical applications of the BPEIs and other biomedical materials.