The MAP65 family of microtubule-associated proteins performs various functions at different stages of the cell cycle and differentiation. In this study, we have investigated the synchronous transdifferentiation of Zinnia mesophyll cells into tracheary elements in vitro. This allowed us to examine the role of the microtubule-associated protein MAP65 during the characteristic bunching of cortical microtubules that underlie the developing ribs of secondarily thickened cell wall. Immunofluorescence confirmed the microtubule bundles to be decorated with anti-MAP65 antibodies. Three Zinnia MAP65 genes were examined; the expression of ZeMAP65-1 was found to match that of the differentiation marker TED2 and both were found to be upregulated upon addition of inductive hormones. We cloned the full-length sequence of ZeMAP65-1 and found it to be most similar to other MAP65 isoforms known to bundle microtubules in other plant species. However, not all MAP65 proteins crosslink cortical microtubules and so, to confirm its potential bundling capacity, ZeMAP65-1 was transiently overexpressed in Arabidopsis suspension cells. This resulted in the super-bundling of microtubules in patterns resembling those in differentiating xylem cells. These findings establish that the MAP65-1 group of proteins is responsible for the bundling of cortical microtubules during secondary cell wall formation of xylogenesis as well as during the expansion of primary cell walls.