AtMAP65-1 gene was cloned from Arabidopsis genome. A plant expression vector containing AtMAP65-1-GFP fusion gene driven by 35S promoter was constructed and introduced into Arabidopsis by Agrobacterium infection. Under confocal laser scanning microscope (CLSM), it was found that in the light-induced opening guard cells AtMAP65-1 was distributed radiately from dorsal wall to ventral wall in a bundling way. Treated with microtubule specific inhibitor-Vinblastine (10 micromol/L) for 90 min, the organization of AtMAP65-1 in guard cell was disrupted dramatically. Following the depolymerization of cortical microtubule and the decreasing of stomatal aperture, the fluorescence of AtMAP65-1 became to be fragmented into dotted spots. The organization of AtMAP65-1 was broken up completely when the treatment was up to 120 min. In the dark-induced closing guard cells, AtMAP65-1 was turned into fragments and was randomly dispersed throughout the cells. That is, AtMAP65-1 is co-localized with cortical microtubules. The results suggest that AtMAP65-1 binds cortical microtubules, and it may regulate the arrangement of cortical microtubules, which helps us gain more insights into the role of AtMAP65-1 in stomatal movement.