In cells, motor proteins do not work alone but rather move in ensemble when they support several cellular functions including mitosis and beating of flagellum. Until now, collective transportation by motors was poorly understood due to the experimental difficulty in controlling the number and arrangement of motors, which are considered to affect the transportation. In this report, we propose a novel nano-patterning method to control the number of kinesin molecules and the spacing between molecules, which transport a single microtubule filament. We fabricated Au nano-pillar array on Si/SiO2 substrates. The SiO2 surface was coated with a silane-poly(ethylene glycol) (PEG) self-assembled monolayer (SAM) using a silane-coupling reaction to eliminate non-specific adsorption of kinesin molecules. By designing the arrangement of pillars, the number of kinesin molecules and the spacing between molecules can be defined, and their effects on kinesin motility was investigated. Two motor species with different processivity, kinesin-1 and Ncd, were patterned individually. We found the number of motors affected the microtubule velocity in the case of non-processive Ncd, but not in the case of processive kinesin-1.