The mechanical characteristics of human red blood cell (RBC) membrane change due to C60 nanoparticle (NP) infiltration have been investigated in the present work. Using experimental approaches, including optical tweezer (OT) stretching and atomic force microscopy (AFM) indentation, we found that RBCs in the presence of C60 NPs are softer than normal RBCs. The strain–stress relations of both normal and C60 infiltrated RBC membranes are extracted from the data of AFM indentation, from which we proved that C60 NP infiltration can affect the mechanical properties of RBC membrane and tend to weaken the tensile resistance of lipids bilayers. In order to explain this experimental phenomenon, a mechanical model has been developed. Based on this model, the strain–stress relations of both normal and C60 infiltrated lipid bilayers are calculated with consideration of intermolecular interactions. The theoretical results are in great agreement with the experimental results. The influence of C60 NP concentration on the mechanical properties of RBC membrane is successfully predicted. Higher concentrations of C60 NPs in the lipid bilayers will lead to increased damage to the cell membrane, implying that the dosage of C60 NPs should be controlled in medical applications.