Both estrogen receptor (ER) positive breast cancer cells MCF-7 and ER-negative MDA-MB-231 cells were employed. Icaritin inhibited the proliferation and migration, and induced cell cycle arrest of both MDA-MB-231 and MCF7 cells. Our study demonstrated that the antitumor effects of icaritin against breast cancer are related with ER, which suggested that the status of ER should be considered in clinical application of icaritin.