The EB1 family proteins are highly conserved microtubule-associated proteins. The EB1 protein in yeast has been shown to play an important role in regulating microtubule dynamics and chromosome segregation. Human EB1 family proteins include EB1, RP1 and EBF3. Although EB1 and RP1 have been shown to associate with microtubules, the subcellular localization of endogenous EBF3 had not been characterized. The function of human EB1 family proteins was also not clear. We therefore investigated the cellular localization of EBF3 and the regulation of microtubule organization by EB1 family proteins. As do EB1 and RP1, EBF3 was found to colocalize with microtubules, preferentially at their plus ends, throughout the cell cycle. Moreover, there was a very strong EBF3 signal at the centrosome in interphase cells and at the spindle poles in mitotic cells. When EB1 family proteins were overexpressed, they associated with the entire microtubule cytoskeleton. In addition, EB1 and EBF3 induced microtubule bundling in some cells overexpressing these proteins. These microtubule bundles were more resistant to nocodazole and were more acetylated than regular microtubules. Our results demonstrate for the first time that human EB1 family proteins could regulate microtubule assembly and stability.