Tubulin, as a crucial structural component of microtubules, is a target for the development of anticancer agents.In this paper, a series of millepachine derivants were synthesized and evaluated for their in vitro and in vivo antiproliferative activity.Among these novel derivatives, compound 50 and 66 dispayed potent antiproliferative activity against various kinds of tumor cell lines including multidrug resistant phenotype.Furthermore, flow cytometry studies showed them arrested the cells in G2/M phase distinctly.Tubulin assembly studies showed that, unexpectly, 66 stabilized tubulin polymerization similar to the paclitaxel while 50 inhibited tubulin polymerization like colchicine.