Parthenolide is an active sesquiterpene lactone present in a variety of medicinal herbs, well known as anti-inflammatory drug. It has recently been proposed as a chemotherapeutic drug, but the pharmacological pathways of its action have not yet been fully elucidated. Firstly, we explored whether the anticancer properties of parthenolide may be related to a tubulin/microtubule-interfering activity. We additionally compared bioactivities of parthenolide with those checked after combined treatments with paclitaxel in human breast cancer MCF-7 cells. Parthenolide exerted in vitro stimulatory activity on tubulin assembly, by inducing the formation of well-organized microtubule polymers. Light microscopy detections showed that parthenolide-induced alterations of either microtubule network and nuclear morphology happened only after combined exposures to paclitaxel. In addition, the growth of MCF-7 cells was significantly inhibited by parthenolide, which enhanced paclitaxel effectiveness. In conclusion, the antimicrotubular and antiproliferative effects of parthenolide, well known microtubule-stabilizing anticancer agent, may influence paclitaxel activity. The tubulin/microtubule system may represent a novel molecular target for parthenolide, to be utilized in developing new combinational anticancer strategies.