© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Biomolecular motors are the smallest natural machines that can convert chemical energy into mechanical work with much higher energy efficiencies compared to man-made machineries. Nowadays, reconstructed biomolecular motors, such as microtubules–kinesin, are successfully utilized for nanotechnological applications, e.g., in nanotransportation, parallel computation, molecular robotics, and so on. However, stochastic nature of their motion poses a limitation to their applications, which is difficult to control particularly under spatial constraints. In this work, top-down and bottom-up approaches are combined to address this problem in a gliding assay of microtubules. Through mechanical stimulation of the motile microtubule filaments, parallelization of the filaments is demonstrated concurrently in hundreds of microwells. The orientation of plenty of motile microtubules in confined space should further accelerate nanotechnological applications of biomolecular motors.