We have demonstrated in vitro motility assay of microtubules on a kinesin-coated substrate by using adenosine triphosphate (ATP) generated from microsphere gels containing thylakoid membranes. Upon photoirradiation, the gels generated ATP, which was utilized for performing gliding motions of microtubules. This work may facilitate the development of a localized ATP generation system in the in vitro motility assay, which consequently would widen the applications of biomolecular motors in nanotechnology.