Activation of heregulin (HRG) signaling has been implicated in the development of aggressive phenotype in breast cancer cells. The mechanisms through which HRG regulates the progression of breast cancer cells to a more invasive or motile phenotype are currently unknown. Because the process of cell migration must involve dynamic changes in the formation of new focal adhesions at the leading edge and dissolution of preexisting focal points, we explored the potential HRG regulation of paxillin, a major component of focal adhesion. Here, we report that HRG stimulation of noninvasive breast cancer MCF-7 cells resulted in the up-regulation of paxillin mRNA and protein. The observed HRG stimulation of paxillin mRNA expression was completely blocked by actinomycin D (a transcriptional inhibitor) as well as by cycloheximide (a protein synthesis inhibitor), suggesting the involvement of an inducible protein factor(s) and transcriptional regulation of paxillin mRNA by HRG. Extension of these observations to other HRG-responsive human cell lines also demonstrated that HRG has a significant capacity to up-regulate the paxillin expression. Furthermore, the levels of paxillin expression were closely linked with the coexpression of human epidermal growth factor receptor 2 (HER2)/HER3 receptors in breast cancer cell lines and in grade III human breast tumors. This study is the first demonstration of regulation of paxillin expression by a polypeptide growth factor, and it suggests a potential role for paxillin in the HER2 pathway in breast cancer.