A novel series of per-O-acetyl-β-D-thoi-glycoside modified shikonin derivatives were designed and synthesized and their anti-proliferative activities were evaluated in vitro on three cancer cell lines. The half maximal inhibitory concentration (IC50) of the mono-, di- and tri- substituted shikonin derivatives vary from 1.58±0.17μg mL-1 to 16.72 ± 1.66μg mL-1. Notably, compared with the parent drug shikonin (IC50 = 2.12 ± 0.17μg mL-1, 6.80 ± 0.21μg mL-1, 5.18 ± 0.69μg mL-1 against MG63, MCF-7 and B16-F10, respectively), IIb (IC50 = 1.04 ± 0.22μg mL-1, 2.02 ± 0.16μg mL-1 and 3.86 ± 0.24μg mL-1) displayed much stronger anti-proliferative effect among them. In addition, IIb exhibited the most potent anti-tubulin activity (IC50 of 3.98 ± 0.43μg mL-1), which compared with shikonin (IC50 = 4.86 ± 0.12μg mL-1). Docking simulation was performed for the position of IIb into the tubulin active site to determine the probable binding model. And the result showed that IIb could bind to the colchicines’ binding site of tubulin nicely. Thus IIb is expected to be the candidate of anti-cancer agent.