Dynein motor proteins usually work as a group in vesicle transport, mitosis, and ciliary/flagellar beating inside cells. Despite the obvious importance of the functions of dynein, the effect of inter-dynein interactions on collective motility remains poorly understood due to the difficulty in building large dynein ensembles with defined geometry. Here, we describe a method to build dynein ensembles to investigate the collective motility of dynein on microtubules. Using electron microscopy, we show that tens to hundreds of cytoplasmic dynein monomers were anchored along a 4- or 10-helix DNA nanotube with an average periodicity of 19 or 44 nm (a programmed periodicity of 14 or 28 nm, respectively). They drove the sliding movement of DNA nanotubes along microtubules at a velocity of 170-620 nm/s. Reducing the stiffness of DNA nanotubes made the nanotube movement discontinuous and considerably slower. Decreasing the spacing between motors simply slowed down the nanotube movement. This slowdown was independent of the number of motors involved but heavily dependent on motor-motor distance. This suggests that steric hindrance or mechanical coupling between dynein molecules was responsible for the slowdown. Furthermore, we observed cyclical buckling of DNA nanotubes on microtubules, reminiscent of ciliary/flagellar beating. These results highlight the importance of the geometric arrangement of dynein motors on their collective motility. (C) 2020 Elsevier Inc. All rights reserved.