In this work, the anticancer activity of chamaejasmin A was studied by evaluating its in vitro cytotoxicity against several cell lines (CAL-27, UMSCC-1, UMSCCG19, HEP-2 and Vero cells) using the 3-(4,5)-dimethylthiazoly1)-3,5-diphenytetrazolium bromide assay. Results indicated chamaejasmin A shows more notable anticancer activity against HEP-2 cells, with IC(50) values of 3.48 μM. Furthermore, western blot analysis showed that chamaejasmin A is able to increase the expression of β-tubulin (TB), but not α-TB. In vivo, chamaejasmin A intake through gavage resulted in β-TB depolymerization inhibition in HEP-2 tumors. In silico simulations indicated that chamaejasmin A specifically interacts with the binding site which is located at the top of β-TB, thanks to the presence of strong hydrophobic effects between the core templates and the hydrophobic surface of the TB protein active site, associated with two strong H-bonds. The binding energy (E (inter)) was calculated to be -129.40 kcal mol(-1). Results above suggest that chamaejasmin A possesses anti-cancer properties relating to β-TB depolymerization inhibition.