Class III β-tubulin (β3) is associated with tumor aggressiveness, resistance to therapy, and patient relapse. To elucidate its action, we tested β3's effect on cell migration. Expression of β3 in HeLa and MCF-7 did not alter the intrinsic rate of cell migration, but it prevented the inhibition of migration by low, nontoxic concentrations of paclitaxel. The effects on cell motility were confirmed in CHO cells with tetracycline regulated expression of β3. Cell migration and microtubule dynamics were inhibited by similar concentrations of paclitaxel, but required a 5-10 fold higher drug concentration when β3 was expressed. The directionality of migration was normal in paclitaxel, but cells spent more time in a "paused" state during which there was no net movement. These studies support a model in which paclitaxel inhibits cell migration by suppressing microtubule dynamics and β3-tubulin counteracts paclitaxel action by maintaining microtubule dynamic activity. The results provide a potential explanation for the aggressiveness of β3-expressing tumors.