Alk(en)yl sulfides are characteristic flavor components of garlic. Several lines of epidemiological study indicate that the risk of a certain cancer can be prevented by consumption of garlic. In this manuscript, we examined the anticancer property of garlic-derived alk(en)yl sulfides, and the molecular basis especially for diallyl trisulfide which is a major constituent of the garlic oil. Alk(en)yl sulfides with different numbers of sulfur atom (i.e., mono-, di-, and trisulfide) were synthesized and purified (>99%). The anticancer activity of the alk(en)yl sulfides was primarily examined using human colon cancer cells HCT-15 and DLD-1. The growth of the cells was significantly suppressed by diallyl trisulfide, but neither diallyl monosulfide nor diallyl disulfide showed such an effect. The number of cells arrested at G2/M phase, the cells with a sub-G1 DNA content, and the cells with caspase-3 activity were dramatically increased by diallyl trisulfide treatment. Diallyl trisulfide disrupted microtubule network formation of the cells, and microtubule fragments could be seen at the interphase. There was a specific oxidative modification of cysteine residues Cys12 beta and Cys354 beta, forming S-allylmercaptocysteines in the tubulin molecule. These results suggest that diallyl trisulfide is responsible, at least in part, for the epidemiologically proven anticancer effect for garlic eaters.