A new series of pyrano chalcone derivatives containing indole moiety (3-42, 49a-49r) were synthesized and evaluated for their antiproliferative activities. Among all the compounds, compound 49b with a propionyloxy group at the 4-position of the left phenyl ring and N-methyl-5-indoly on the right ring displayed the most potent cytotoxic activity against all tested cancer cell lines including multidrug resistant phenotype, which inhibits cancer cell growth with IC50 values ranging from 0.22 to 1.80μM. Furthermore, 49b significantly induced cell cycle arrest in G2/M phase and inhibited the polymerization of tubulin. Molecular docking analysis demonstrated the interaction of 49b at the colchicine binding site of tubulin. In experiments in vivo, 49b exerted potent anticancer activity in HepG2 human liver carcinoma in BALB/c nude mice. These results indicated these compounds are promising inhibitors of tubulin polymerization for the potential treatment of cancer.