Microtubules are highly dynamic cytoskeletal polymers of α/β-tubulin heterodimers that undergo multiple post-translational modifications essential for various cellular functions in eukaryotes. The lysine 40 (K40) is largely conserved in α-tubulins in many eukaryote species, and the post-translational modification by acetylation at K40 is critical for neuronal development in vertebrates. However, the biological function of K40 of α-tubulins in plants remains unexplored. In this study, we show in Arabidopsis thaliana that constitutive expression of mutated forms of α-tubulin6 (TUA6) at K40 (TUA6(K40A) or TUA6(K40Q) ), in which K40 is replaced by alanine or glutamine, result in severely reduced plant size. Phenotypic characterization of the 35S:TUA6(K40A) transgenic plants revealed that both cell proliferation and cell expansion were affected. Cytological and biochemical analyses showed that the accumulation of α- and β-tubulin proteins was significantly reduced in the transgenic plants, and the cortical microtubule arrays were severely disrupted, indicating that K40 of the plant α-tubulin is critical in maintaining microtubule stability. We also constructed 35S:TUA6(K40R) transgenic plants in which K40 of the engineered TUA6 protein is replaced by an arginine, and found that the 35S:TUA6(K40R) plants were phenotypically indistinguishable from the wild-type. Since lysine and arginine are similar in biochemical nature but arginine cannot be acetylated, these results suggest a structural importance for K40 of α-tubulins in cell division and expansion.