HER2/neu, a receptor tyrosine kinase oncogene, promotes mitogenic growth and transformation of cancer cells. We previously identified that its oncogenic signals down-regulate the cyclin-dependent kinase inhibitor p27 Kip1, which is defined as a haplo-insufficient tumor suppressor. Here, we applied the human p27 gene as a novel anticancer agent for HER2/neu-overexpressing cells under the control of a tetracycline (tet)-regulated gene expression system. Overexpression of p27 inhibits HER2/neu-activated CDK2 activity, cell proliferation, and transformation. Most significantly for clinical application, p27 expression in HER2/neu-overexpressing cells can be regulated in vivo and reduce the tumor volume in a tumor model. The findings demonstrate the applicability of employing p27 in HER2/neu-associated cancer gene therapy.