BACKGROUND Most breast cancers are estrogen dependent and were sensitive to endocrine therapy, and genistein (GEN) shows strong affinity with human oestrogen receptor beta (ERβ). OBJECTIVE The present study aimed to investigate the anticancer activity of GEN in breast cancer cell lines that constitutively expressing ERβ1 in vitro and in vivo. METHODS MCF-7/ERβ1 and MDA-MB-231/ERβ1 cell sub-lines were established through lentiviral infection. Then, cells were treated with increasing concentrations of GEN (10-6 mol/l, 10-5 mol/l and 10-4 mol/l) for 48 h, and cell proliferation, cell cycle analyses were performed to investigate different biological characteristics of ERβ1-overexpressing cell lines. Studies in vivo were also performed to investigate the effects of dietary GEN on MCF-7/ERβ1 and MDA-MB-231/ERβ1 cells implanted mice. RESULTS Results showed that compared to parental cells, GEN inhibited the proliferation ability of MCF-7/ERβ1 cells to a greater extent, especially at high concentrations. MDA-MB-231 cells were also inhibited by high doses of GEN, but the overexpressed ERβ1 did not enhance the anti-proliferative effect on MDA-MB-231 cells. ERβ1 arrested cells in G2/M phase, and GEN arrested cells in G0/G1, which led to a combinatorial effect on cell cycle blockade. Furthermore, ERβ1 increased the anti-tumour activity of dietary GEN in MCF-7/ERβ1 subcutaneous tumour models. Our data indicated that ERβ1 increased the anticancer efficacy of GEN in MCF-7 cells by affecting cell cycle transition. CONCLUSIONS As a result, GEN could be a potential therapeutic agent for ERβ1-positive cancer.