Objective To investigate the effects of brusatol on mechanical properties of the cytoskeleton as well as the invasion behavior of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). Methods Cytoskeleton staining method was used to determine the regulatory effects of brusatol on mechanical properties of the RA FLS cytoskeleton. Transwell chamber assay was used to detect the effects of brusatol on the cytoskeleton and invasion behavior of RA FLS. The effects of brusatol on the expression of matrix metalloproteinase-2/3 (MMP-2/3) of RA FLS were measured by zymography and Western blotting methods. Results Cytoskeleton staining and microscope observation showed that brusatol could significantly reduce the formation number of RA FLS pseudopodia, thus inhibited cell movement ability via regulating mechanical properties of cytoskeleton. The invasion behavior of RA FLS was inhibited by brusatol, and brusatol could down-regulate the expression of MMP-2/3. Conclusions Brusatol plays an important role in regulating mechanical properties of cytoskeleton and inhibiting the invasion behavior of RA FLS. Meanwhile, brusatol can inhibit the invasion behavior of RA FLS through down-regulating the expression of MMP-2/3. The research findings provide the corresponding experimental basis for further development of new drugs for RA treatment.