Natural products are a major source of anticancer agents and play critical roles in anticancer drug development. Inspired by the complexity-to-diversity strategy, novel deoxypodophyllotoxin (DPT) analogues were designed and synthesized. Among them, compound <b>C3</b> exhibited the potent antiproliferative activity against four human cancer cell lines with IC<sub>50</sub> values in the low nanomolar range. Additionally, it showed marked activity against paclitaxel-resistant MCF-7 cells and A549 cells. Moreover, compound <b>C3</b> can inhibit tubulin polymerization by targeting the colchicine-binding site of tubulin. Further study revealed that compound <b>C3</b> could arrest cancer cells in the G<sub>2</sub>/M phase and disrupt the angiogenesis in human umbilical vein endothelial cells. Meanwhile, <b>C3</b> remarkably inhibited cancer cell motility and migration, as well as considerably inhibited tumor growth in MCF-7 and MCF-7/TxR xenograft model without obvious toxicity. Collectively, these results indicated that compound <b>C3</b> may be a promising tubulin polymerization inhibitor development for cancer treatment.