The present data showed that a novel synthesized compound, 6-acetyl-9-(3,4,5-trimethoxybenzyl)-9H-pyrido [2,3-b]indole (HAC-Y6), exhibited potent antitumor activity against human hepatocellular carcinoma (HCC) cells in vitro. Western blot and immunofluorescence experiments showed that HAC-Y6 depolymerized microtubules similarly to the effects of colchicine. HAC-Y6-treatment in Hep3B cells resulted in the accumulation of the G2/M phase and induced apoptosis. In addition, HAC-Y6-treatment influenced the expression of cell cycle and apoptosis related proteins in Hep3B cells. HAC-Y6 exposure increased caspases-3, -8, -9 and Bax protein levels, while reducing levels of Bcl-2 family proteins. Moreover, Bid, a substrate of caspase-8, was also activated by HAC-Y6. Treatment of cells caused the up-regulation of the death receptor 4 (DR4) and phosphorylation of p38. Taken together, we show that HAC-Y6 exhibited its antitumor activity by disrupting microtubule assembly, causing cell cycle arrest and apoptosis through both extrinsic and intrinsic pathways in Hep3B cells. Therefore, the novel compound HAC-Y6 is a promising microtubule inhibitor that has great potential for treatment of HCC.