This paper reports an evaluation of microtubules gliding characteristics on surface roughness structures, which were fabricated on polyimide films by reactive ion etching. Surface roughness was increased with increase of etching time, and the maximum surface roughness was approximately 116 nm at 20 min etching time. In the comparison of gliding velocities on the fabricated films and on glass substrate, there was significant difference at the surface roughness of over 82 nm. Over 70% of microtubules stopped on the fabricated films with the surface roughness of over 42 nm. These results showed that the surface roughness structure influences the characteristics of microtubules gliding, and it could be useful for a trapping-structure in gliding assay.