Twenty-five novel pyranochalcone derivatives were synthesized and evaluated for their in vitro and in vivo antiproliferative activities. Among them, compound 10i exhibited superior potent activity against 21 tumor cell lines including multidrug resistant phenotype with the IC50 values ranged from 0.09 to 1.30 μM. In addition, 10i significantly induced cell cycle arrest in G2/M phase, promoted tubulin polymerization into microtubules and caused microtubule stabilization. Further studies confirmed that 10i significantly suppressed the growth of tumor volume in HepG2 xenograft tumor model. Our study demonstrated that 10i could have beneficial antitumor activity as a novel microtubule stabilizing agent.