Triphala (Trl) is an ayurvedic formulation used for treating disorders of the digestive, respiratory, and nervous systems. Its anticancer properties have also been documented. We studied effects of Trl on tubulin, a target protein for several anticancer drugs, and systematically elucidated a possible antiproliferative mechanism of action of Trl. Trl inhibited proliferation of HeLa (cervical adenocarcinoma), PANC-1 (pancreatic adenocarcinoma), and MDA-MB-231 (triple-negative breast carcinoma) cells in microgram quantities and strongly suppressed the clonogenicity of HeLa cells. The formulation disrupted secondary conformation of tubulin and inhibited anilino naphthalene sulfonate binding to tubulin. In cells, Trl-tubulin interactions were manifested as a perturbed microtubule network. Acetylation pattern of Trl-treated cellular microtubules indicated persistent stabilization of microtubule dynamics. In addition, Trl interfered with reassembly of the microtubules. Cells treated with Trl eventually underwent programmed cell death as evidenced by annexin-V staining. Our study shows that the effect of aqueous extract of Trl is potent enough to interfere with the assembly dynamics of microtubules, and that Trl can be investigated further for its antitumor potential.