We propose a nano-patterning method to control the number of kinesin motors involving in a collective transport of a single microtubule (MT) filament. We fabricatedAu nano-pillar array on a thermally oxidized silicon substrate. SiO2 surface was selectively coated with poly(ethylene glycol) (PEG) self-assembled monolayer (SAM) and was rendered inert to protein adsorption. Streptavidin was immobilized only on Au nano-pillars to immobilize kinesin-1 motors. MTs glided on the top of kinesin-1-immobilized Au nano-pillars, where the number of motors and the spacing between motors transporting a single MT were defined. Our patterning method helps to study how the collective dynamics of multiple motors are regulated by the number of motors and the arrangement of motors with an in vitro assay.