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In vitro gliding assay, microtubule translocation by kinesin motor proteins on a surface, has been used as an engineering tool in analyte detection, molecular cargo transport, and other applications. Although controlling the moving direction is often necessary to realize these applications, current direction control methods focus largely on lithographic microfabrication of tracks or external fields on the microtubules. These methods are effective, but are relatively complicated. In addition, they cannot target particular microtubules without affecting others. In this study, we propose a facile approach that can make local direction changes for selected microtubules using a polystyrene particle as a circular motion center and a DNA double helix with streptavidin as a capture arm. The DNA arm captures a microtubule in the close proximity of the immobilized particle via biotin–streptavidin interaction and changes the moving direction ~10° on average. In contrast, no significant direction changes are observed other than random variations with streptavidin‐less DNA arms (normal distribution centered at 0°), similar to regular motility assay. The particle‐assisted local direction change scheme is compared with a flow field‐based ensemble method. The combination of flow and kinesin interactions with each microtubule exerts a force to change the direction, ultimately aligning it to the flow field, regardless of its initial direction. A simple model based on the force balance predicts the time needed for such an alignment. Overall, the particle‐based local scheme is distinct and different from ensemble methods such as crossflow that changes directions of all microtubules in the field, thus offering unique utility in engineering applications.