Understanding what modulates the cell stiffness is important given its potential application as a diagnostic and medical target. Here, we investigated why and how mono-fullerenols affect the cell stiffness. We confirmed the fullerenol-modulation of cell stiffness using atomic force microscopy (AFM) with sphere tips and ascertained that the particles reduce the cell polarity. The structures of b-actin and f-actin were evaluated by inverted fluorescence microscopy, synchrotron radiation small angle X-ray scattering (SAXS), transmission electron microscopy (TEM) and AFM. Statistical and quantitative analyses of the SAXS data of fullerenol-treated b-actin and f-actin reveal a transformation from large-size to small-size b-actin and simultaneously to f-actin. The slight increase in f-actin diameter in the treated group suggests that fullerenols attach to the actin surface. We verified the attachment using AFM and high-resolution probes. Collectively, our results suggest that fullerenols hamper the bundling of f-actin to form b-actin by adhering to the surface of f-actin, weakening the bundle-based cell stiffness.