Estrogen receptors are activated by the hormone estrogen and they control cell growth by altering gene expression as a transcription factor. So far two estrogen receptors have been found: ERα and ERβ. Estrogen receptors are also implicated in the development and progression of breast cancer. Here, we found that ERα localized on the spindle and spindle poles at the metaphase during mitosis. Depletion of ERα generated unaligned chromosomes in metaphase cells and lagging chromosomes in anaphase cells in a transcription-independent manner. Furthermore, the levels of β-tubulin and γ-tubulin were reduced in ERα-depleted cells. Consistent with this, polymerization of microtubules in ERα-depleted cells and turnover rate of α/β-tubulin were decreased than in control cells. We suggest that ERα regulates chromosome alignment and spindle dynamics by stabilizing microtubules during mitosis.