Soy isoflavone genistein, a tyrosine kinase inhibitor and agonist of estrogen receptor-β (ERβ), is known to have antitumoral properties. Given that ERβ often is coexpressed with HER2 in breast cancer, both functions of genistein might be able to enhance the antitumoral action of trastuzumab. In this in-vitro study, we tested whether combined treatment with genistein and trastuzumab exerts additive effects on breast cancer cells. HER2-overexpressing breast cancer cell lines were treated with genistein alone and in combination with trastuzumab. The effects of this treatment on proliferation and gene expression were analyzed. Treatment with high-dose genistein (10 μmol/l) significantly increased the growth-inhibitory effect of trastuzumab on HER2-overexpressing, ERα/β-positive BT-474 breast cancer cells. Combinatory treatment using lower doses of trastuzumab exerted similar effects as a single treatment with standard doses of this drug. In contrast, this effect was absent in ERα-negative SK-BR-3 cells. Similar results were obtained after cotreatment with the ERβ agonist, 2,3-bis(4-hydroxyphenyl)propionitrile. The growth-inhibitory effect of both drugs was accompanied by an increased expression of the putative tumor suppressor ERβ variant, cx, and their combination further elevated mRNA levels of this receptor. In conclusion, genistein significantly enhanced the antitumoral effect of trastuzumab on BT-474 breast cancer cells in vitro. The relevance of these data particularly for women with HER2-overexpressing and ERα/β-positive breast cancer has to be verified in animal or clinical studies.