Gliding microtubules (MTs) on a surface coated with kinesin biomolecular motors have been suggested for the development of nanoscale transport systems. In order to establish a sorting function for gliding MTs, events for MTs approaching micro-scale grooves were investigated. MTs longer than the width of grooves fabricated on a Si substrate bridged the grooves (bridging) and many MTs shorter than the groove width almost began to bridge, but returned to the surface that they approached from (guiding). Occurrence probabilities for the events were analyzed with focus on the geometric conditions, such as length of the MTs, width of the grooves, and the incident angle (α) of the MTs approaching the grooves. The occurrence probability for bridging increased with an increase in the incident angle (16%, α = 0–30°; 51%, α = 30–60°; 75%, α = 60–90°), and the probability for guiding decreased with an increase in the incident angle (79%, α = 0–30°; 55%, α = 30–60°; 5%, α = 60–90°). The results indicate that an incident angle of 30–60° is an effective condition for MT sorting, because the bridging and guiding events can sort MTs that are longer and shorter than the groove widths, respectively. Furthermore, the occurrence probabilities of both bridging and guiding in a higher concentration of methylcellulose (0.5%) increased up to approximately 70% at incident angles of 30–60°, indicating good feasibility for the development of devices for the sorting of MTs on surfaces with topographical grooves.