Background: Cistanche deserticola, a traditional Chinese herb medicine, has been widely used for thousands of years with the activities of hormone regulation, immunomodulatory, antioxidative, neuroprotective, anti-inflammatory, and estrogen. Glycosides of Cistanches (GCs) were the main bioactivity components of the herb. Objective: The objective of the study is to study estrogenic activity and the mechanism about estrogen receptors (ERs) of GCs. Materials and Methods: Cell proliferation was measured using the 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyltetrazolium bromide assay for MCF-7 cells. The cell cycle was detected using flow cytometry, and proliferation index was calculated. The mRNA and protein expressions of ERα and ERβ were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis as the reported method with minor modifications. Results: GCs group at the concentrations of 1.75, 17.5, and 175 μg/mg could enhance proliferation of the MCF-7 cell lines with a time and dosage-dependent manner. Combined medication group (fulvestrant with estradiol [E2] or GCs) could lead to the incline of proliferation rate compared with the individual medication group (P < 0.01). Flow cytometry analysis indicated that GCs could advance MCF-7 cell lines from G0/G1 phase cells to S and G2/M phase, which could promote cell DNA synthesis. The mechanism of GCs on MCF-7 was similar to that of E2. RT-PCR and western blot analysis indicated that after treatment with GCs for 48 h, contents of ERα and ERβ mRNA and proteins in MCF-7 increased as a dosage-dependent manner with that of GCs. GCs can play a role of estrogenic activity according upregulated mRNA and proteins of ERα and ERβ. Conclusion: This study indicated the estrogenic activity of GCs, and also, ER is the target of GCs. GCs can play a role of estrogenic activity according upregulated mRNA and proteins of ERα and ERβ.