Post-translational modifications (PTMs) of ?/?-tubulin are believed to regulate interactions with microtubule-binding proteins. A well-characterized PTM involves in the removal and re-ligation of the C-terminal tyrosine on ?-tubulin, but the purpose of this tyrosination-detyrosination cycle remains elusive. Here, we examined the processive motility of mammalian dynein complexed with dynactin and BicD2 (DDB) on tyrosinated versus detyrosinated microtubules. Motility was decreased ~fourfold on detyrosinated microtubules, constituting the largest effect of a tubulin PTM on motor function observed to date. This preference is mediated by dynactin's microtubule-binding p150 subunit rather than dynein itself. Interestingly, on a bipartite microtubule consisting of tyrosinated and detyrosinated segments, DDB molecules that initiated movement on tyrosinated tubulin continued moving into the segment composed of detyrosinated tubulin. This result indicates that the ?-tubulin tyrosine facilitates initial motor-tubulin encounters, but is not needed for subsequent motility. Our results reveal a strong effect of the C-terminal ?-tubulin tyrosine on dynein-dynactin motility and suggest that the tubulin tyrosination cycle could modulate the initiation of dynein-driven motility in cells.