Newly translated tubulin molecules undergo a series of complex interactions with nascent chain-binding chaperones, including prefoldin (PFD) and chaperonin-containing TCP-1 (CCT). By screening for oryzalin hypersensitivity, we identified several mutants of Arabidopsis that have lesions in PFD subunits. The pfd6–1 mutant exhibits a range of microtubule defects, including hypersensitivity to oryzalin, defects in cell division, cortical array organization, and microtubule dynamicity. Consistent with phenotypic analysis, proteomic analysis indicates several isoforms of tubulins were reduced in pfd6–1 . These results support the concept that the function of microtubules is critically dependent on the absolute amount of tubulins.