Cytoskeleton nanomechanics characterizes cancer cell's physical behaviors such as how it spread and invade. For anticancer drug, cytoskeleton nanomechanics may be a target to inhibit invasiveness and metastasis of cancer cells. Therefore, in vitro assay of cytoskeleton nanomechanics may be used to evaluate the effects of potential anticancer drug on various cancer types. Here, we investigated the effects of tubeimoside I (TBMS I) on human hepatoma (HepG2) cells by using optical magnetic twisting cytometry, a well-established technique for measuring nanomechanics of the Factin cytoskeleton. TBMS I is a natural compound extracted from a traditional Chinese herbal medicine, and is reported with antitumor effect. In this study, we demonstrated that the cytoskeleton stiffness (G) of HepG2 cells was affected by TBMS I. G′ exhibited a typical power law with respect to the loading frequency (f), i.e. G^f . The magnitude of G′ and the value of exponent (α) of the HepG2 cells decreased consistently with the increase of concentration for TBMS I exposure. In addition, the HepG2 cells responded to TBMS I much faster than the normal liver (L-02) cells. Such alteration of cytoskeleton nanomechanics induced by TBMS I was reported for the first time, which was further corroborated by assays of Factin cytoskeleton structure and cell migration. Taken together, these results suggest that in vitro assay of cytoskeleton nanomechanics may have a great potential as an additional tool in screening of anticancer drug candidates.