Background: Estrogen receptor beta (ER beta) is expressed in the majority of invasive breast cancer cases, irrespective of their subtype, including triple-negative breast cancer (TNBC). Thus, ER beta might be a potential target for therapy of this challenging cancer type. In this in vitro study, we examined the role of ER beta in invasion of two triple-negative breast cancer cell lines. Methods: MDA-MB-231 and HS578T breast cancer cells were treated with the specific ER beta agonists ERB-041, WAY200070, Liquiritigenin and 3 beta-Adiol. Knockdown of ER beta expression was performed by means of siRNA transfection. Effects on cellular invasion were assessed in vitro by means of a modified Boyden chamber assay. Transcriptome analyses were performed using Affymetrix Human Gene 1.0 ST microarrays. Pathway and gene network analyses were performed by means of Genomatix and Ingenuity Pathway Analysis software. Results: Invasiveness of MBA-MB-231 and HS578T breast cancer cells decreased after treatment with ER beta agonists ERB-041 and WAY200070. Agonists Liquiritigenin and 3 beta-Adiol only reduced invasion of MDA-MB-231 cells. Knockdown of ER beta expression increased invasiveness of MDA-MB-231 cells about 3-fold. Transcriptome and pathway analyses revealed that ER beta knockdown led to activation of TGF beta signalling and induced expression of a network of genes with functions in extracellular matrix, tumor cell invasion and vitamin D3 metabolism. Conclusions: Our data suggest that ER beta suppresses invasiveness of triple-negative breast cancer cells in vitro. Whether ER beta agonists might be useful drugs in the treatment of triple-negative breast cancer, has to be evaluated in further animal and clinical studies.