In this study, the alterations of the morphology and biomechanical properties of living SMCC-7721 cancer cells treated with fullerenol (C60(OH)24) for 24, 48, and 72h were investigated using an atomic force microscope (AFM). Comparative analyses show that the elastic moduli of the SMCC-7721 cells exposed to fullerenol decrease significantly with the increase of the treatment periods. Furthermore, in different phases of the treatment, a global decrease in elasticity is accompanied by cellular morphological changes, and the time-dependent effect of the fullerenol can be observed using AFM and optical microscope. In addition, as the treatment duration increases, the indentation force and depth penetrated into the cell membrane by the AFM tip are in a declining trend. The reduction in the stiffness of the cells exposed to fullerenol could be associated with the disruption of the cellular cytoskeleton network. The investigation indicates that the elastic modulus of single living cells can be a useful biomarker to evaluate the effects of fullerenol or other anticancer agents on the cells and reveal instructive information for cellular dynamic behaviors.