Expressed in the human breast and in human breast cancer tissues, the heterodimeric peptide hormone relaxin is involved in extracellular matrix turnover. To investigate the role of relaxin in estrogen receptor-alpha negative human breast cancer cells, we established transfectants of the human MDA-MB-231 breast cancer cell line stably overexpressing H2-relaxin (MDA-MB-231/pIRES-EGFP-H2). These transfectants produced and secreted functional relaxin. Our investigations revealed downregulation of mRNA and protein of the calcium binding protein S100A4 (metastasin) in MDA-MB-231/pIRES-EGFP-H2 transfectants, indicating a distinct and novel role for relaxin in estrogen receptor-alpha negative human breast cancer cells.